CN108785669A

CN108785669A - Antibody preparation and application thereof

Info

Publication number: CN108785669A
Application number: CN201810659773.9A
Authority: CN
Inventors: S.贝内迪特; M.C.曼宁; B.M.墨菲; S.里尔; C.朔伊尔
Original assignee: TRACON
Current assignee: TRACON
Priority date: 2012-09-05
Filing date: 2013-09-05
Publication date: 2018-11-13
Also published as: AU2013312536B2; CA2883343A1; EP2892559A4; AU2013312536A1; MX368996B; HK1210714A1; JP6445671B2; SG11201501659SA; AU2018204044A1; EA029214B1; CN104684580A; KR101820582B1; JP2018076350A; UA115789C2; KR20180007014A; EP2892559A1; SG10201705070PA; PH12015500480A1; GEP201706789B; JP2015532657A

Abstract

This application involves anti-CD105 antibody, the preparations and application thereof of its antigen-binding fragment.It is related to the precharging injection syringe of anti-CD105 antibody or the preparation of its antigen-binding fragment on the other hand.It is related to the purposes of one or more S or Ss of the preparation for reducing angiogenesis associated disease such as cancer and ophthalmology disease on the other hand.

Description

Antibody preparation and application thereof

It is on 09 05th, 2013 that the application, which is the applying date, application No. is 201380046405.2, entitled " antibody The divisional application of the application for a patent for invention of preparation and application thereof ".

This application claims the equity for the U.S. Provisional Application No. 61/697,111 that September in 2012 is submitted on the 5th, and this application is with it It is incorporated herein by reference.This application involves following co-pending patent applications：US publication US 2010- 0098692 A1 [attorney number 35882-706.201]；8,221,753 [attorney number of U.S. Patent number 35882-706.202]；U.S. Application No. 13/485,702 [attorney number 35882-706.301]；And U.S. Application No. 13/390,896 [attorney number 35882-712.201], is incorporated herein by reference with it.

Sequence table

The application contains via EFS-Web with ASCII fromat submission and herein with it entirely through the sequence being incorporated by Table.The ASCII copies generated, entitled 35882-714.601_SL.txt, 22,121 word of size on the 4th in September in 2013 Section.

Technical field

This application involves anti-CD105 antibody, the preparations and application thereof of its antigen-binding fragment.It is related to resisting on the other hand The precharging injection syringe of the preparation of CD105 antibody or its antigen-binding fragment.It is related to the preparation on the other hand for reducing The purposes of one or more S or Ss of angiogenesis associated disease such as cancer and ophthalmology disease.

Background technology

Cancer is to be only second to the second largest human death's reason of coronary artery disease.In the world, there is millions of people dead every year In cancer.Only in the U.S., cancer is caused every year more than 500,000 people death, and has about 1,400,000 cases newly made a definite diagnosis every year.Although Because deaths from heart disease has been decreased obviously, dead caused by cancer is typically to increase.In many countries, cancer has been most The main cause of the death.

In addition, even for those of their primary cancer cancer patient is initially spent, common experience has confirmed, Their service life drastically changes.Many cancer patients can undergo strong anxiety, and the anxiety is by recurrence or treatment failure Possibility knows to drive.Many cancer patients undergo significant in poor health after treatment.

In general, the root problem of the control of mortality cancer is, lack effective and nontoxic constitutional treatment.Cancer It is a kind of disease of complexity, it is characterised in that lead to the genetic mutation of cell growth out of control.Cancer cell is present in all biologies In body, under normal circumstances, their undue growth by different physiological factors close adjusting.

Angiogenesis is the physiology course from existing vascularization new blood vessel.It has been proposed that angiogenesis is in normal processes With all work in pathologic process.For example, angiogenesis is related to the development of the vascular system of animal organ and tissue.

Under certain pathological states, angiogenesis is stimulated, as the blood for providing abundance for the cell in affected tissue The mode of liquid and nutrient supply.Pathological states as many include abnormal cell Proliferation and/or adjusting.It solid carcinoma and oozes Going out property macular degeneration depends on raises new blood supply for continuous growth and transfer.

Invention content

There is provided herein the novel formulation of anti-CD105 antibody, the precharging injection syringe containing the preparation and such preparations Purposes for treating angiogenesis associated disease.This application provides can be used for the application of intravenous or intraocular, for example, treatment with The preparation of CD105 relevant cancer and ophthalmic conditions.

In one aspect, there is provided herein include the anti-CD105 antibody of about 1 mg/ml to about 150 mg/ml or its antigen knot Close segment, the preparation of at most about 100 mM buffers, at most about 1 M polyalcohols and the pH of about 4.0 to about 7.5.

In one aspect, the preparation is stable after preparation, can be tested according to usual manner.With regard to the system It is such as measured by size exclusion chromatography method (SEC) for agent is with the stability of time, at least 95% anti-CD105 antibody Or its antigen-binding fragment stores at about 2 to 8 DEG C and monomer can be used as to exist after at least about 12 months.In some embodiments In, the buffer of such preparation is histidine or phosphate buffered saline (PBS).For the preparation is with the stability of time, Such as measured by size exclusion chromatography method (SEC), at least 90% anti-CD105 antibody or its antigen-binding fragment are at about 25 DEG C Storage can be used as monomer to exist after at least about 6 months.In some embodiments, the buffer of such preparation is acetic acid Salt.

In addition, stored at about 2-8 DEG C after at least about 12 months can be with for the anti-CD105 antibody or its antigen-binding fragment Display about 50 to about 150% combines (passing through CD105 ELISA binding assays).

After being stored at least about 12 months at 2 to 8 DEG C, the average isoelectric point (pI) of anti-CD105 antibody can be about 8.7 to about 9.2, as by, for example, measured by the electricity focusing such as Capillary Electrophoresis.

It will be appreciated by those skilled in the art that anti-CD105 antibody or its antigen-binding fragment can stablize at least about 12 months, At least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months or longer time.

After the freezing of the preparation and thaw cycle, as measured by SEC, the preparation can contain as single At least 95% anti-CD105 antibody existing for body.Alternatively, or additionally, when being subjected to agitation stress, as passed through SEC institutes It measures, preparation can contain the anti-CD105 antibody as existing for monomer at least 95%.

Anti- CD105 antibody or its antigen-binding fragment can include can be in conjunction with any sequences of CDR of CD105.At one In non-limiting embodiments, the anti-CD105 antibody includes to have such as SEQ ID NO:The light chain of amino acid sequence shown in 1 can Become area (V_L)；With such as SEQ ID NO:Constant region of light chain (the C of amino acid sequence shown in 2_L)；With such as SEQ ID NO:Shown in 3 Heavy chain variable region (the V of amino acid sequence_H)；With with such as SEQ ID NO:Constant region (the Fc) (ginseng of amino acid sequence shown in 4 See, for example, Fig. 1).

In another non-limiting embodiment, the anti-CD105 antibody includes to have such as SEQ ID NO:Ammonia shown in 5 The V of base acid sequence_LCDR1；With such as SEQ ID NO:The V of amino acid sequence shown in 6_LCDR2；With such as SEQ ID NO:7 institutes Show the V of amino acid sequence_LCDR3；With such as SEQ ID NO:The V of amino acid sequence shown in 8_HCDR1；With such as SEQ ID NO:The V of amino acid sequence shown in 9_HCDR2；With with such as SEQ ID NO:The V of amino acid sequence shown in 10_H CDR3。

The immune anti-CD105 antibody that goes of the humanization of separation can include to have such as SEQ ID NO:Amino acid shown in 11 The heavy chain variable region of sequence and with such as SEQ ID NO:The light chain variable region of amino acid sequence shown in 12.Humanization-goes to be immunized Other non-limiting examples of heavy chain include, but are not limited to SEQ ID NO:13,14,15 and 16.Humanization-removes immune light chain Other non-limiting examples include, but are not limited to SEQ ID NO:17,18,19,20,21,22 and 23.Sequence is provided below In embodiment 17.

The preparation contains about 1 mg/ml to the anti-CD105 antibody of about 150 mg/ml or its antigen-binding fragment, or Any of which value, including but not limited to, about 2 mg/ml, about 5 mg/ml, about 7.5 mg/ml, about 10 mg/ml, 20 mg/ml, About 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, About 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, About 95 mg/ml, about 100 mg/ml, about 105 mg/ml, about 110 mg/ml, about 115 mg/ml, about 120 mg/ml, about 125 Mg/ml, about 130 mg/ml, about 135 mg/ml, about 140 mg/ml, about 145 mg/ml, about 150 mg/ml or more.

In one embodiment, the preparation includes the anti-CD105 antibody or antigen-binding fragment of about 25 mg/ml.

In another embodiment, the preparation includes the anti-CD105 antibody or antigen-binding fragment of about 50 mg/ml.

In another embodiment again, the preparation includes the anti-CD105 antibody or antigen binding fragment of about 100 mg/ml Section.

Preparation as described herein can contain buffer, such as, for example, histidine, acetate, citrate or phosphoric acid Salt.In one embodiment, the preparation includes about 5 mM, about 7.5 mM, about 10 mM, about 12.5 mM, about 15 mM, about 17.5 mM, 20 mM, about 22.5 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 MM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM or about 100 mM Histidine, acetate, citrate or phosphate.

Preparation can prepare any kind of application for becoming known for antibody, include, but are not limited in vitreum and Intravenous application.

Preparation provided herein may further include acceptable carrier or excipient, including as pharmaceutically acceptable Carrier or excipient and for being applied to the acceptable any carrier of patient or excipient.

In one embodiment, preparation provided herein is isotonic." isotonic " expression target formulation has and people's blood The identical osmotic pressure of liquid-based sheet.Isotonic preparation will usually have the osmotic pressure of about 250 to 350 millisomoles (mOsm).Isotonicity can It is (for example) measured using vapour pressure or ice-frozen type osmometer.In another embodiment, preparation provided herein is Hypertonic." isotonic " preparation is the preparation with the osmotic pressure essentially identical with human blood.Correspondingly, term is " hypertonic " is used for retouching State the preparation with the osmotic pressure higher than human blood.

Preparation provided herein contains the polyalcohol of the amount less than 1 M.For example, polyalcohol can about 50 mM, about 75 MM, about 100 mM, about 150 mM, about 200 mM, about 225 mM, about 240 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 MM, about 850 mM, about 900 mM, about 950 mM, about 1 M or in which any integer amount be present in the preparation.At one In embodiment, the preparation is made into isotonic, and wherein salinity is about 100 mM to about 175 mM.For example, containing being less than The preparation of the polyalcohol of 300 mM amounts is made into isotonic, and wherein salinity is about 130 mM.

In one aspect, the polyalcohol being ready to use in preparation provided herein can be sugar, such as, for example, irreducibility Sugar.The representative example of nonreducing sugar includes, but are not limited to trehalose and sucrose.For example, the preparation can include about 200 MM is to about 300 mM trehaloses or sucrose.In one embodiment, preparation can include about 240 mM trehaloses or sucrose.

Alternatively, the sugar can be the D-sorbite of the amount (concentration) of about 200 mM to about 300 mM.In an embodiment party In case, preparation can include about 240 mM D-sorbites.

Other non-limiting examples of preparation provided herein are any one of preparation 1-39 of table 1.

Preparation provided herein can be with the pH of about 4.0 to about 7.5.In one embodiment, preparation provided herein It can be with about 4.5, about 5.0, about 5.5, about 6.0, about 6.5 or about 7.0 pH.

In one embodiment, preparation provided herein does not contain surfactant.Optionally, in some cases, Surfactant may include in the formulation.The non-limiting examples of surfactant include polysorbate 20, poly- sorb Sugar alcohol ester 80 and Pluronic F68.

It is also provided herein and is suitable for intravenous or intravitreal administration pre-filled note comprising preparation as described herein Emitter.Such precharging injection syringe can be packaged and mark for treating the relevant situation of angiogenesis, such as any Situation described in text.Packaging can further comprise the instruction for storing and applying.There is provided herein include preceding claims Any one of preparation the packaging for being suitable for intravenous or intravitreal administration one or more precharging injection syringes.

The embodiment of the application considers that any composition as described herein is used for the purposes of compounding pharmaceutical, institute Drug is stated for treating illness as described herein.Pharmacy can be matched based on the physical trait of patient/subject in need for the treatment of Object, and single preparation or multiple preparations can be configured to.Can by drug packages in the suitable package with appropriate label with Hospital and clinic are distributed to, wherein the label is indicated for the illness as described herein for the treatment of subject.Drug can wrap Dress up single or multiple units.It may include the specification of the dosage and application about composition described herein in packaging.

There is provided herein the methods for treating the relevant disease of angiogenesis in patient (subject) in need comprising Preparation as described herein is applied to the patient.Such preparation can be in vitreum or to be intravenously applied to patient.

The relevant disease of angiogenesis as described herein can be, for example, cancer or transfer.In one embodiment, The cancer is solid tumor.Cancer to be treated includes, for example, the tumour based on epithelium.The cancer of for use such preparation for treating The non-limiting examples of disease include, but are not limited to lung cancer, gynecologic malignant tumor, melanoma, breast cancer, cancer of pancreas, ovary Cancer, uterine cancer, colorectal cancer, prostate cancer, kidney, head cancer, cancer of pancreas, liver cancer (hepatocellular carcinoma), uterine cancer, neck Cancer, kidney (clear-cell carcinoma), sarcoma, myeloma and lymthoma.It can intravenously be applied for treating cancer or the preparation of transfer For patient.

Alternatively, the relevant disease of angiogenesis as described herein can be, for example, ophthalmic conditions.Ophthalmic conditions include, but It is not limited to, age-dependent macular degeneration, diabetic retinopathy, macular edema and/or choroidal neovascularization are formed. Age-dependent macular degeneration (AMD) can be moist AMD or dryness AMD.Preparation for treating ophthalmic conditions can be with glass It is applied to patient in vivo.

In such method, the preparation one or many can be applied to patient.For example, the preparation can be daily Once, once a week, monthly, every month twice, each two moon is primary, every three months is primary, every four months it is primary, every 5 months primary or every 6 months applied onces.Therapeutic scheme can as needed increase according to the response of patient for treatment to add deduct It is few.

In one aspect, using preparation, until one or more S or Ss of the relevant disease of angiogenesis mitigate.

For ophthalmic conditions, one or more S or Ss may include, but are not limited to vasoconstriction, inhibit with The relevant endothelial cell proliferation of eye disease, the muddy eyesight for the treatment of, provides stopping for visual loss at the S or S for removing bleeding Stagnant, improvement eyesight improves visual acuity, reduces macular edema and/or prevents vascular leakage.

For cancer or transfer, treatment can lead to the improvement of status of patient, and treatment can pass through the following factor of determination One or more of whether have occurred and that evaluate：The cell Proliferation of reduction, the cell number of reduction, increased cell wither It dies or the survival of the cell of at least part formation cell proliferative disorders reduces.

Treatment can lead to the tumour of patient or the partially or completely elimination of transfer and/or prolonging survival.

In one embodiment, after the preparation to the one or more dosage of patient's application, one or more signs Symptom severity or the duration reduce about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, About 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 95% or about 100%.

In another embodiment, after the preparation to the one or more dosage of patient's application, one or more bodies Sign or symptom severity or the duration reduce about 2 times, about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 Times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 90 Again, about 95 times, about 100 times or more times.

There is provided herein the methods for treating the ophthalmic conditions in patient in need comprising herein to patient application The preparation, thus one or more S or Ss of the ophthalmic conditions are treated mitigates.The application of the preparation can To be intravitreal administration.

The method that prevention or cancer or transfer in treatment subject in need is also provided herein comprising to described Patient applies preparation as described herein, and thus the cancer or one or more S or Ss of transfer mitigate.The preparation Application can intravenously apply.

In method provided herein, subject to be treated can be people or non-human subject.Preparation provided herein It can be using the effect of one or many, this depends on the health of patient, the progress of disease or situation and treatment.It was treating Cheng Zhong can adjust therapy and the treatment dosage of antibody (for example, in composition).

There is provided herein methods the effect of monitoring any one or more methods provided herein.Soluble CD105 It is horizontal related to the survival of cancer patient, and can be monitored before treatment with period.Therefore, soluble The level of CD105 can be a kind of instruction that therapeutic scheme effectively treats patient.Treatment as described herein may include one kind Or a variety of additional treatments.

It is incorporated by reference into

The all publications, patents and patent applications referred in the present specification are all herein incorporated by reference, and degree is such as With pointing out specifically and that individually every individual publication, patent or patent application are incorporated by reference into.

Therefore, the present invention provides the specific implementation modes described in the following terms：

1. preparation, it includes the anti-CD105 antibody of about 1 mg/ml to about 150 mg/ml or its antigen-binding fragment, at most about 100 The pH of mM buffers, at most about 1 M polyalcohols and about 4.0 to about 7.5.

2. 1 preparation, wherein such as measured by size exclusion chromatography method (SEC), at least 95% anti-CD105 antibody exists About 2 to 8 DEG C of storages exist as monomer at least 12 months later.

3. 1 preparation, wherein such as measured by size exclusion chromatography method (SEC), at least 95% anti-CD105 antibody exists Exist as monomer after about 25 DEG C of storage at least six moons.

4. 1 preparation, wherein such as measured by size exclusion chromatography method (SEC), at least 90% anti-CD105 antibody exists Exist as monomer after about 25 DEG C of storage at least six moons.

5. 1 preparation, wherein the buffer is histidine or phosphate buffered saline (PBS).

6. 1 preparation, wherein the buffer is acetate, and pH is about 4.

7. 1 preparation, wherein the buffer is histidine, and pH is about 5.5.

8. 1 preparation, wherein the anti-CD105 antibody or its antigen-binding fragment store at least 12 months at about 2 to 8 DEG C Display later is combined by about the 50 to 150% of CD105 ELISA binding assays.

9. 1 preparation, wherein as measured by Capillary Electrophoresis-isoelectric focusing, stored at least 12 months at 2 to 8 DEG C Later, the average isoelectric point (pI) of the anti-CD105 antibody is about 8.7 to about 9.2.

10. 1 preparation, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:Amino acid sequence shown in 1 it is light Chain variable region (V_L)；With such as SEQ ID NO:Constant region of light chain (the C of amino acid sequence shown in 2_L)；With such as SEQ ID NO:3 Heavy chain variable region (the V of shown amino acid sequence_H)；With with such as SEQ ID NO:The constant region (Fc) of amino acid sequence shown in 4.

11. 1 preparation, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:The V of amino acid sequence shown in 5_L CDR1；With such as SEQ ID NO:The V of amino acid sequence shown in 6_LCDR2；With such as SEQ ID NO:Amino acid sequence shown in 7 V_LCDR3；With such as SEQ ID NO:The V of amino acid sequence shown in 8_HCDR1；With such as SEQ ID NO:Amino acid shown in 9 The V of sequence_HCDR2；With with such as SEQ ID NO:The V of amino acid sequence shown in 10_H CDR3。

12. 1 preparation, it includes the anti-CD105 antibody of about 25 mg/ml or its antigen-binding fragments.

13. 1 preparation, it includes the anti-CD105 antibody of about 50 mg/ml or its antigen-binding fragments.

14. 1 preparation, it includes the anti-CD105 antibody of about 100 mg/ml or its antigen-binding fragments.

15. 1 preparation, wherein the buffer is histidine or acetate.

16. 15 preparation, it includes about 20 mM histidines or acetates.

17. 1 preparation, wherein the preparation is formulated in vitreum or intravenous application.

18. 1 preparation, further includes acceptable carrier or excipient.

19. 18 preparation, wherein the carrier or excipient are pharmaceutically acceptable carrier or excipient.

20. 1 preparation, is isotonic or hypertonic.

21. 1 preparation, wherein the polyalcohol is less than 300 mM, and the preparation be made of salt it is isotonic.

22. 1 preparation, wherein after the freezing of the preparation and thaw cycle, as measured by SEC, at least 95% anti-CD105 antibody exists as monomer.

23. 1 preparation, wherein when being subjected to agitation stress, as measured by SEC, at least 95% anti-CD105 antibody Exist as monomer.

24. 1 preparation, wherein the polyalcohol is sugar.

25. 24 preparation, wherein the sugar is non-reducing sugar.

26. 25 preparation, wherein the nonreducing sugar is trehalose or sucrose.

27. 26 preparation, it includes about 240 mM trehaloses or sucrose.

28. 24 preparation, wherein the sugar is D-sorbite.

29. 28 preparation, it includes about 240 mM D-sorbites.

30. 1 preparation, further includes surfactant.

31. 30 preparation, wherein the surfactant is polysorbate20, polysorbate80 or Pluronic F68。

32. being suitable for intravenous or intravitreal administration precharging injection syringe comprising any one of aforementioned preparation.

33. the method for the angiogenesis-associated diseases in treatment subject in need comprising to the patient apply comprising The anti-CD105 antibody of about 1 mg/ml to about 150 mg/ml or its antigen-binding fragment, at most about 100 mM buffers, at most about 1 The preparation of M polyalcohols and the pH of about 4.0 to about 7.5.

34. 33 method, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:Amino acid sequence shown in 1 it is light Chain variable region (V_L)；With such as SEQ ID NO:Constant region of light chain (the C of amino acid sequence shown in 2_L)；With such as SEQ ID NO:3 Heavy chain variable region (the V of shown amino acid sequence_H)；With with such as SEQ ID NO:The constant region (Fc) of amino acid sequence shown in 4.

35. 33 method, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:The V of amino acid sequence shown in 5_L CDR1；With such as SEQ ID NO:The V of amino acid sequence shown in 6_LCDR2；With such as SEQ ID NO:Amino acid sequence shown in 7 V_LCDR3；With such as SEQ ID NO:The V of amino acid sequence shown in 8_HCDR1；With such as SEQ ID NO:Amino acid shown in 9 The V of sequence_HCDR2；With with such as SEQ ID NO:The V of amino acid sequence shown in 10_H CDR3。

36. 33 method, it includes the anti-CD105 antibody of about 25 mg/ml or its antigen-binding fragments.

37. 33 method, it includes the anti-CD105 antibody of about 50 mg/ml or its antigen-binding fragments.

38. 33 method, it includes the anti-CD105 antibody of about 100 mg/ml or its antigen-binding fragments.

39. 33 method, wherein the preparation includes to be less than 300 mM polyalcohols, and the preparation be made of salt it is isotonic.

40. 33 method, wherein the buffer is histidine or acetate.

41. 40 method, it includes about 20 mM histidines or acetates.

42. 33 method, wherein the polyalcohol is sugar.

43. 42 method, wherein the sugar is nonreducing sugar.

44. 43 method, wherein the nonreducing sugar is trehalose or sucrose.

45. 44 method, it includes about 240 mM trehaloses or sucrose.

46. 42 method, wherein the sugar is D-sorbite.

47. 46 method, it includes about 240 mM D-sorbites.

48. 33 method wherein applies the preparation in vitreum or intravenously.

49. 33 method, further includes surfactant.

50. 49 method, wherein the surfactant is polysorbate20, polysorbate80 or Pluronic F68。

51. 33 method, wherein the angiogenesis-associated diseases are cancer or transfer.

52. 51 method, wherein the cancer is solid tumor.

53. 51 method, wherein the cancer is the tumour based on epithelium.

54. 51 method, wherein the cancer is lung cancer, gynecologic malignant tumor, melanoma, breast cancer, cancer of pancreas, ovum Nest cancer, uterine cancer, colorectal cancer, prostate cancer, kidney, head cancer, cancer of pancreas, liver cancer (hepatocellular carcinoma), uterine cancer, neck Portion's cancer, kidney (clear-cell carcinoma), sarcoma, myeloma, the cancer of the brain or lymthoma.

55. 33 method, wherein the angiogenesis-associated diseases are ophthalmology diseases.

56. 55 method, wherein the ophthalmic conditions be age-dependent macular degeneration, diabetic retinopathy or Choroidal neovascularization is formed.

57. 56 method, wherein the age-dependent macular degeneration (AMD) is moist AMD or dryness AMD.

58. 33 method, wherein it is one or many to apply the preparation to the patient.

59. 33 method, wherein once a day, once a week, monthly, every month twice, each two moon it is primary, Every three months is primary, preparation described in primary, every 5 months every four months primary or every 6 months applied onces.

60. 33 method, wherein the preparation is applied, until one or more signs of the angiogenesis-associated diseases Or symptom mitigates.

61. 60 method, wherein the severity of one or more S or Ss or duration reduce about 2%, About 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, About 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 95% or about 100%.

62. 60 method, wherein the severity of one or more S or Ss or duration reduce about 2 times, About 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, About 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 90 times, about 95 times, about 100 times or more times.

63. 60 method, wherein one or more S or Ss are that vasoconstriction, inhibition are related to eye disease Endothelial cell proliferation, remove bleeding S or S, the muddy eyesight for the treatment of, the stagnation that visual loss is provided, improve eyesight And/or prevent vascular leakage.

64. 33 method, wherein described treat the improvement for leading to status of patient, and can be by the following factor of determination Whether one or more has occurred and that evaluate：The cell Proliferation of reduction, the cell number of reduction, increased Apoptosis or The survival that at least part forms the cell of cell proliferative disorders reduces.

65. 33 method, wherein treatment leads to the tumour of patient or the partially or completely elimination of transfer and/or prolonging survival.

66. the method for the ophthalmic conditions in treatment patient in need comprising applied to the patient and include about 1 mg/ml To the anti-CD105 antibody of about 150 mg/ml or its antigen-binding fragment, at most about 100 mM buffers, at most about 1 M polyalcohols, The preparation of the pH of about 4.0 to about 7.5 and optional surfactant, thus one or more signs of the ophthalmic conditions or disease Shape is eased.

67. 66 method, wherein the ophthalmic conditions be age-dependent macular degeneration, diabetic retinopathy or Choroidal neovascularization is formed.

68. 67 method, wherein the age-dependent macular degeneration (AMD) is moist AMD or dryness AMD.

69. 66 method, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:Amino acid sequence shown in 1 it is light Chain variable region (V_L)；With such as SEQ ID NO:Constant region of light chain (the C of amino acid sequence shown in 2_L)；With such as SEQ ID NO:3 Heavy chain variable region (the V of shown amino acid sequence_H)；With with such as SEQ ID NO:The constant region (Fc) of amino acid sequence shown in 4.

70. 66 method, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:The V of amino acid sequence shown in 5_L CDR1；With such as SEQ ID NO:The V of amino acid sequence shown in 6_LCDR2；With such as SEQ ID NO:Amino acid sequence shown in 7 V_LCDR3；With such as SEQ ID NO:The V of amino acid sequence shown in 8_HCDR1；With such as SEQ ID NO:Amino acid shown in 9 The V of sequence_HCDR2；With with such as SEQ ID NO:The V of amino acid sequence shown in 10_H CDR3。

71. 66 method, wherein one or more S or Ss are that vasoconstriction, inhibition are related to eye disease Endothelial cell proliferation, remove bleeding S or S, the muddy eyesight for the treatment of, the stagnation that visual loss is provided, improve eyesight And/or prevent vascular leakage.

72. 66 method, wherein the severity of one or more S or Ss or duration reduce about 2%, About 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, About 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 95% or about 100%.

73. 66 method, wherein the severity of one or more S or Ss or duration reduce about 2 times, About 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, About 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 90 times, about 95 times, about 100 times or more times.

74. 66 method, wherein the preparation includes to be less than 300 mM polyalcohols, and the preparation be made of salt it is isotonic.

75. preventing or the method for the treatment of the cancer or transfer in subject in need comprising to the patient apply comprising The anti-CD105 antibody of about 1 mg/ml to about 150 mg/ml or its antigen-binding fragment, at most about 100 mM buffers, at most about 1 The preparation of M polyalcohols and the pH of about 4.0 to about 7.5, thus the cancer or one or more S or Ss of transfer obtain Alleviate.

76. 75 method, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:Amino acid sequence shown in 1 it is light Chain variable region (V_L)；With such as SEQ ID NO:Constant region of light chain (the C of amino acid sequence shown in 2_L)；With such as SEQ ID NO:3 Heavy chain variable region (the V of shown amino acid sequence_H)；With with such as SEQ ID NO:The constant region (Fc) of amino acid sequence shown in 4.

77. 75 method, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:The V of amino acid sequence shown in 5_L CDR1；With such as SEQ ID NO:The V of amino acid sequence shown in 6_LCDR2；With such as SEQ ID NO:Amino acid sequence shown in 7 V_LCDR3；With such as SEQ ID NO:The V of amino acid sequence shown in 8_HCDR1；With such as SEQ ID NO:Amino acid shown in 9 The V of sequence_HCDR2；With with such as SEQ ID NO:The V of amino acid sequence shown in 10_H CDR3。

78. 75 method, wherein the application of the preparation extends the life of the subject.

79. 75 method, wherein the cancer is primary tumor or metastatic tumo(u)r.

80. 75 method, wherein the cancer is solid tumor.

81. 80 method, wherein the solid tumor is the solid tumor of tissue or organ selected from the following：Skin, melanin Tumor, lung, pancreas, breast, ovary, colon, rectum, stomach, thyroid gland, throat, ovary, prostate, colorectum, head, neck, Eye, mouth, larynx, oesophagus, chest, bone, testis, lymph, marrow, bone, sarcoma, kidney, sweat gland, liver, kidney, brain (such as pleomorphism colloid Blastoma, glioma) etc..

82. 81 method, wherein the solid tumor is colon tumor, tumor of breast, kidney neoplasms, lung neoplasm, forefront adenoncus The transfer of tumor, ovarian neoplasm or any such tumour.

83. 75 method, wherein described treat the improvement for leading to subject's situation, and can be by the following factor of determination One or more whether have occurred and that evaluate：The cell Proliferation of reduction, the cell number of reduction, increased Apoptosis, Or at least part forms the survival reduction of the cell of cell proliferative disorders.

84. 75 method, wherein treatment leads to the tumour of patient or the partially or completely elimination of transfer and/or prolonging survival.

85. 75 method, wherein the severity of one or more S or Ss or duration reduce about 2%, About 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, About 60%, about 65%, about 70%, about 75%, about 80%, about 90%, about 95% or about 100%.

86. 75 method, wherein the severity of one or more S or Ss or duration reduce about 2 times, About 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, About 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 90 times, about 95 times, about 100 times or more times.

87. 75 method, it includes the anti-CD105 antibody of about 25 mg/ml or its antigen-binding fragments.

88. 75 method, it includes the anti-CD105 antibody of about 50 mg/ml or its antigen-binding fragments.

89. the method for claim 75, it includes the anti-CD105 antibody of about 100 mg/ml or its antigen-binding fragments.

90. 75 method, wherein the buffer is histidine or acetate.

91. 90 method, it includes about 20 mM histidines or acetates.

92. 75 method, wherein the polyalcohol is sugar.

93. 92 method, wherein the sugar is nonreducing sugar.

94. 93 method, wherein the nonreducing sugar is trehalose or sucrose.

95. 94 method, it includes about 240 mM trehaloses or sucrose.

96. 92 method, wherein the sugar is D-sorbite.

97. 96 method, it includes about 240 mM D-sorbites.

98. 75 method, wherein the preparation includes to be less than 300 mM polyalcohols, and the preparation be made of salt it is isotonic.

99. 75 method, further includes surfactant.

100. 99 method, wherein the surfactant is polysorbate20, polysorbate80 or Pluronic F68。

101. 75 method, wherein intravenously applying the preparation.

102. preparation, it includes any one of preparation 1-39 of table 1.

103. precharging injection syringe, it includes the preparations of item 102.

104. 102 preparation is used to treat the purposes of angiogenesis-associated diseases.

105. purposes of 102 preparation in preparing the drug for treating angiogenesis-associated diseases.

Description of the drawings

The new feature of embodiment has been described in detail in the appended claims.By reference to describing illustrative embodiment party Case (principle for wherein using embodiment) and the feature and advantage described below that can obtain the present embodiment of attached drawing It is best understood from, the attached drawing is as follows：

Figure 1A-J provide the exemplary amino acid sequence of anti-CD105 antibody (TRC105) as described herein.Figure 1A is anti-CD105 The representativeness of antibody can lighten (VL) chain (SEQ ID NO:1)；Figure 1B is the representative constant light (CL) of anti-CD105 antibody (SEQ ID NO:2)；Fig. 1 C are representative Weight variable (VL) chain (SEQ ID NO of anti-CD105 antibody:3)；And Fig. 1 D are anti- Constant γ -1 heavy chains (the SEQ ID NO of representativeness of CD105 antibody:4).Fig. 1 E-G respectively represent the CDR 1,2 and 3 of VL.Fig. 1 H- J respectively represents the CDR 1,2 and 3 of VH.

Fig. 2 provides the schematic diagram of TGF-β/ALK5 signal transduction paths.TGF-β/ALK5 approach (A) leads to cell Proliferation Inhibit.TGF-β/ALK1 approach (B) inducing cell proliferation, and CD105 (endothelial factor) is needed to carry out ALK1 signal transductions. Dotted line indicates inactive or blocking approach.The stimulation of the arrow indication signal pathway of overstriking.

Detailed description of the invention

It should be appreciated that terminology used in this article is only used for the purpose of description specific embodiment, it is not intended to be limited.This Outside, it should be understood that many methods and material similar or equivalent with those described herein can be used for putting into practice the present invention.

According to the application, conventional cell biology, molecular biology, microbiology and recombinant DNA technology may be used, As this field is explained completely.

As used in this specification and appended claims, singulative " one (a) ", " one (an) " and " being somebody's turn to do (the) " Including plural reference, clearly indicate unless the context otherwise.Thus, for example, referring to that " method " includes being described herein and/or readding After reader disclosure those skilled in the art will become apparent from one or more methods and/or step of the type Suddenly.

Anti- CD105 antibody can be used to treat or prevent the relevant illness of various forms of angiogenesis.This document describes logical Cross the method for treating or preventing various forms of cancers, solid tumor and transfer etc. using preparation described herein.

Antibody term

Terms used herein " antibody " indicate immunoglobulin (Ig), no matter natural or partially or even wholly synthesis life Production.The term is also covered by any more peptide or proteins with binding structural domain, and the binding structural domain is antigen binding structure Domain is homologous with antigen-binding domains.The term also comprises " antigen-binding fragment " and all similar binding fragments described as follows Other interchangeable terms.

Natural antibody and native immunoglobulin typically about 150, the heterologous tetramer glycoprotein of 000 dalton, by 2 A identical light (L) chain and 2 identical heavy (L) chain compositions.Each light chain usually passes through a covalent disulfide bonds and heavy chain phase Connect, and the number of disulfide bond has differences between the heavy chain of different Immunoglobulin Isotypes.Each heavy chain and light chain also have The intrachain disulfide bond of regular distribution.Each heavy chain has the variable domains (" V an end_H" or " VH "), thereafter It is many constant domain (" C_H" or " CH ").Each light chain has the variable domains (" V an end_L" or " VL ") With the constant domain (" C of another end at it_L" or " CL ")；The constant domain of light chain and the first of heavy chain perseverance Constant domain is aligned, and light variable domains are aligned with the variable domains of heavy chain.Think that particular amino acid residue is formed Interface between light chain and heavy-chain variable domains.

Terms used herein " polynucleotides of synthesis ", " gene of synthesis " or " polypeptide of synthesis " indicate, corresponding Polynucleotide sequence or part thereof or amino acid sequence or part thereof are originated from the sequence designed, or from new synthesis, or with etc. The naturally occurring sequence of effect is compared and is modified.By methods known in the art, the polynucleotides (antibody of synthesis can be prepared Or antigen-binding fragment) or synthesis gene, the method includes but be not limited to：The chemical synthesis of nucleic acid or amino acid sequence. The gene of synthesis is usually different from naturally occurring gene in amino acid or polynucleotide level (or in two levels), and usually In the background of the expression control sequence of synthesis.Compared with natural gene, the gene polynucleotides sequence of synthesis may not Certain albumen of the coding with different aminoacids；For example, they can also include the polynucleotide sequence of synthesis, the multinuclear glycosides Acid sequence mixes different codons, but they encode identical amino acid (that is, nucleotide variation is represented in amino acid levels Silent mutation).

About antibody, term " variable domains " indicates, the combination in each specific antibodies and its specific antigen and spy The constant region for immunoglobulin sequence used in the opposite sex.But variability is unevenly distributed in the variable domains of antibody.On the contrary, It concentrates on 3 and is referred to as in the section of hypervariable region (also referred to as CDR), and the section is in both light chain and heavy-chain variable domains In.The more highly conserved part of variable domains is referred to as " framework region " or " FR ".Unmodified heavy chain and light chain can be changed Structural domain respectively contains 4 FR (FR1, FR2, FR3 and FR4), they mainly take beta sheet configuration, are studded with 3 therebetween CDR, the CDR form ring, and connect beta sheet structure division in some cases.CDR in each chain keeps close by FR Be close together, and together with from the CDR of other chains, promote the antigen binding site of antibody formation (referring to Kabat et al., Sequences of Proteins of Immunological Interest, the 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991), the 647-669 pages).

Terminology used in this article " hypervariable region " and " CDR " expression, the amino acid residue of the responsible antigen binding of antibody. CDR includes the amino acid residue from 3 sequence areas, and the sequence area combines antigen in complementary fashion, and referred to as CDR1, CDR2 and CDR3 are (for each V_HAnd V_LChain).According to Kabat et al., Sequences of Proteins of Immunological Interest, the 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), in light variable domains, CDR generally corresponds to substantially residue 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3), in heavy-chain variable domains, it is residual that CDR generally corresponds to substantially residue Base 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3).It is inserted into it should be appreciated that the CDR of different antibodies can contain Sequence, thus amino acid number may be different.Kabat numbering systems describe such insetion sequence with following numbering plans： The scheme utilize be connected with specific residue letter (for example, in light chain, 27A, 27B, 27C, 27D, 27E of CDRL1 and 27F) it is reflected in any insertion of the number between different antibodies.Alternatively, according to Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)), in light variable domains, CDR generally corresponds to substantially residue 26-32 (CDRL1), 50-52 (CDRL2) and 91-96 (CDRL3), and in heavy-chain variable domains, CDR generally corresponds to disable greatly Base 26-32 (CDRH1), 53-55 (CDRH2) and 96-101 (CDRH3).

" framework region " or " FR " used herein indicate that the framework amino acid for forming a part for antigen binding slot or ditch is residual Base.In some embodiments, the Framework residues form ring, and the ring is a part for antigen binding slot or ditch, and in institute Antigen can be contacted or not contact by stating the amino acid residue in ring.Framework region is typically included in the region between CDR.According to Kabat et al., Sequences of Proteins of Immunological Interest, the 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), light chain can In structure changes domain, FR generally corresponds to substantially residue 0-23 (FRL1), 35-49 (FRL2), 57-88 (FRL3) and 98-109, In heavy-chain variable domains, FR generally corresponds to substantially residue 0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3) and 103- 133.As the Kabat numbers above for light chain are discussed, heavy chain also be inserted into (for example, in heavy chain in a similar way by description In, 35A, 35B of CDRH1).Alternatively, according to Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)), in light variable domains, FR generally corresponds to substantially residue 0-25 (FRL1), 33-49 (FRL2) 53-90 (FRL3) and 97-109 (FRL4), in heavy-chain variable domains, FR generally corresponds to substantially residue 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3) and 102-113 (FRH4).

The constant domain (Fc) of antibody does not participate in the combination of antibody and antigen directly, but shows on the contrary different Effector function, such as by the interaction with such as Fc receptors (FcR), antibody participates in the cytotoxicity of antibody dependent. Fc structural domains can also increase the bioavilability of antibody after being administered to patient in the circulating cycle.With mouse Fc domain substitute people Fc structural domains can also reduce HAMA side reactions.

According to the amino acid sequence of the constant domain of their heavy chain, immunoglobulin can be included into different classes Not.There are 5 main immunoglobulin class：IgA, IgD, IgE, IgG and IgM, several in these can further divide At subclass (isotype), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.It is opposite with different classes of immunoglobulin The heavy chain constant domain (Fc) answered is referred to as α, δ, ε, γ and μ respectively.The subunit structure of different classes of immunoglobulin and 3-d modelling is well-known.

It, can be by the antibody from any invertebrate species based on the amino acid sequence of their constant domain " light chain " is included into one of 2 visibly different types (being referred to as " κ " and " λ ").

Term " antigen-binding portion thereof of antibody ", " antigen-binding fragment ", " antigen-binding domains ", " antibody fragment " or " function fragment of antibody " is used to indicate that one or more segments of antibody, the segment to retain specificity interchangeably herein The ability of ground combination antigen.The non-limiting embodiment for the antibody fragment for including in such term includes but is not limited to： (i) Fab segments, i.e., by V_L、V_H、C_LAnd C_H1The monovalent fragment of structural domain composition；(ii)F(ab')₂Segment passes through containing 2 The bivalent fragment of the connected Fab segments of disulfide bond at hinge area；(iii) by V_HAnd C_H1The Fd segments of structural domain composition； (iv) V of the single armed containing antibody_LAnd V_HThe Fv segments of structural domain；(v) dAb segments (Ward et al., (1989) Nature 341:544 546), contain V_HStructural domain；The CDR of (vi) separation.Also comprise in this definition containing single heavy chain and " half " antibody of single light chain.Also include the single-chain antibody of other forms, such as bispecific antibody herein (diabodies)。

By handling Ig with protease (such as pepsin and papain), " F (ab ') can be produced₂" and The part " Fab' ", and F (ab ')₂With Fab' include by near disulfide bond digest immunoglobulin caused by antibody fragment, The disulfide bond is present between the hinge area in each in 2 heavy chains.For example, papain can be present in 2 IgG is cut in the upstream of the disulfide bond between hinge area in each in heavy chain, to generate 2 homologous antibody fragments, In by V_LAnd C_LThe light chain (constant region of light chain) that constitutes and by V_HAnd C_Hγ1The heavy chain fragment that the area (γ 1) is constituted is (in the perseverance of heavy chain Determine in area) it is connected by disulfide bond in their C-terminal region.Each in this 2 homologous antibody fragments is referred to as Fab'. Pepsin also can cut IgG in the downstream of the disulfide bond between the hinge area in each in being present in 2 heavy chains, with production Raw such antibody fragment：The segment that the segment is connected at hinge area wherein less times greater than 2 above-mentioned Fab'.This is anti- Body segment is referred to as F (ab')₂。

First constant domain (C of Fab the segments also constant domain containing light chain and heavy chain_H1).Fab' segments With Fab segments the difference is that, in heavy chain C_HIt is added to several residues at the c-terminus of 1 structural domain, including comes from antibody hinge One or more cysteines in area.Fab'-SH indicates such Fab' herein：Wherein one or more of constant domain A cysteine residues carry free sulfydryl.F(ab')₂Antibody fragment is initially produced as Fab' segments pair, they have Hinge cysteine between them.Other chemical couplings of antibody fragment are also known.

" Fv " indicates the antibody fragment containing intact antigen identification and antigen binding site.The region is by closely non-covalent Or (disulfide bond company has been described in this field to the dimer of a covalently bound heavy chain and light variable domains composition The Fv connect, Reiter et al. (1996) Nature Biotechnology 14:1239-1245).It is each variable in the configuration 3 CDR of structural domain interact, to be limited to V_H-V_LAntigen binding site on the surface of dimer.From each V_HWith V_LThe combination of one or more CDR of chain assigns antibody antigen binding specificity together.For example, it should be appreciated that for example, when transfer To the V of receptor antibody_HAnd V_LWhen on chain or its antigen-binding fragment, CDRH3 and CDRL3 are enough to assign antibody antigen combination specifically Property, and combination, the affinity etc. of any technical testing CDR as described herein combinations can be used.Even if single varistructure Domain (or half of the Fv only comprising 3 CDR to antigentic specificity) also has the ability for identifying and combining antigen, although affine Power may than it is combined with second variable domains when it is lower.In addition, although 2 structural domain (V of Fv segments_LAnd V_H) by list Only gene code can connect them, the connector allows them to that list is made using recombination method by the connector of synthesis A protein chain, wherein V_LAnd V_HIt matches to form monovalent molecule (referred to as scFv (scFv) in area;Bird et al. (1988) Science 242:423-426;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883；With Osbourn et al. (1998) Nat. Biotechnol. 16:778).In " antigen-binding portion thereof " of term antibody, it is also intended to Including such scFv.It can be by any V of specific scFv_HAnd V_LSequence is connected on the areas Fc cDNA or genome sequence, so as to Prepare the expression vector for encoding complete Ig (for example, IgG) molecules or other isotypes.V_HAnd V_LIt can be used for using ubiquitinated Or recombinant DNA technology prepare other segments of Fab, Fv or Ig.

" scFv " or " sFv " antibody fragment includes the V of antibody_HAnd V_LStructural domain, wherein these structural domains are present in individually In polypeptide chain.In some embodiments, the Fv polypeptides are additionally contained in V_HAnd V_LPeptide linker between structural domain, makes SFv can form the desired structure of antigen binding.About the summary of sFv, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore compile Springer- Verlag, New York, the 269-315 pages (1994).

Term " AVIMER " indicates the treatment albumen of a kind of people source, they are unrelated with antibody and antibody fragment, by several A module and reusable binding structural domain is constituted, and the binding structural domain is referred to as A- structural domains (also referred to as A class moulds Block, complement type repetitive sequence or LDL- receptor class A structural domains).They are by the reorganization of external exon and phage display from people Extracellular receptor domain development forms (Silverman et al., 2005, Nat. Biotechnol. 23:1493-1494; Silverman et al., 2006, Nat. Biotechnol. 24:220).Obtained albumen can contain multiple independent combinations Structural domain, compared with single epitope binding protein, the affinity that can show to improve (in some cases, is rubbed less than receiving for they You) and specificity.See, e.g., U.S. Patent Application Publication No. 2005/0221384,2005/0164301,2005/ 0053973 and 2005/0089932,2005/0048512 and 2004/0175756, each is herein with it entirely through reference It is incorporated herein.

Each in known 217 people A- structural domains includes about 35 amino acid (about 4 kDa)；These structural domain quilts Connector separates, and the length of the connector is average 5 amino acid.Natural A-structural domain is quickly and effectively folded into uniformly , the structure stablized, this is mainly by calcium in conjunction with mediating with disulfide bond formation.The apokoinou construction needs the guarantor of only 12 amino acid Keep holder motif.Final result is the single protein chain containing multiple structural domains, and each structural domain represents individual function.It is described Each structural domain of albumen independently combines, and the energy contribution (energetic contributions) of each structural domain is Cumulative.These albumen are referred to as " AVIMERs " from affinity polymer.

Term " bispecific antibody " indicates that the small antibody fragment with 2 antigen binding sites, the segment are included in phase The heavy-chain variable domains (VH) being connected with light variable domains (VL) in homopolypeptide chain (VH-VL).By using too short Do not allow the connector matched between 2 in same chain structural domains, forces the complementary structure of the structural domain and another chain Domain is matched, and generates 2 antigen binding sites.Bispecific antibody is described in more detail below, for example, EP 404,097; WO 93/ 11161；With Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993)。

Antigen-binding polypeptides also include heavy chain homodimer, such as, for example, resisting from camel class (camelids) and shark Body.Camel class and shark antibody include the homodimer of V- samples and 2 chains in C- spline structures domain to (all not having light chain).Cause For the V of the heavy chain homodimer IgG of camel class_HArea not necessarily generates hydrophobic interaction with light chain, in camel class, usually contacts The region in the heavy chain of light chain becomes hydrophilic amino acid residue.The V of heavy chain homodimer IgG_HStructural domain is referred to as V_HHStructural domain.Shark Fish Ig-NAR includes 1 variable domains (being referred to as V-NAR structural domains) and 5 C- samples constant domains (C-NAR structural domains) Homodimer.In camel class, the diversity of antibody all constituents is by V_HOr V_HHCDR 1,2 and 3 in area is determined.White horse with a black mane Hunchbacked V_HHCDR3 in area is characterized in that its relatively long length, 16 amino acid that are averaged (Muyldermans et al., 1994, Protein Engineering7(9): 1129).This is different from the areas CDR3 of the antibody of many other species.Example Such as, mouse V_HCDR3 there are average 9 amino acid.For example, by public in U.S. Patent Application Serial 20050037421 The method opened can prepare camel class-derivative antibody variable region (they maintain the internal diversity of camel class variable region) Library.

Inhuman (for example, mouse) antibody of " chimeric " form includes：Chimeric antibody containing the minmal sequence from inhuman Ig. To the full extent, chimeric antibody is such mouse antibody：Wherein insert at least one of constant region for immunoglobulin (Fc) (at least part of usual human immunoglobulin(HIg)) is divided to substitute mouse Fc.About details, referring to Jones et al.,Nature 321: 522-525 (1986)；Reichmann et al.,Nature332: 323-329 (1988)；And Presta,Curr. Op. Struct. Biol., 2: 593-596 (1992)。

Terms used herein " monoclonal antibody " indicate the antibody that the antibody population of basically homogeneity obtains, that is, remove May with it is micro it is existing may be other than naturally occurring mutation, to constitute the individual antibody of the group be identical.Monoclonal Antibody is high degree of specificity, for single antigen site.In addition, different from conventional (polyclonal) antibody preparation (they May include the different antibodies for different determinants (epitope)), each monoclonal antibody is for the single determinant on antigen. Qualifier " monoclonal " instruction antibody is characterized in that the antibody population of basically homogeneity obtains, and should not be construed as needing by appointing What ad hoc approach produces antibody.For example, monoclonal antibody can by Kohler et al.,Nature256:495 (1975) are first Prepared by the hybridoma method first described, or can pass through recombinant DNA method (see, e.g., U.S. Patent number 4,816,567) To prepare.In certain embodiments, using for example in Clackson et al.,Nature352:624-628 (1991) and Marks et al.,J. Mol. Biol. 222:Technology described in 581-597 (1991), can be from phage antibody library point Separate out monoclonal antibody.

Pass through saturated ammonium sulphate, the euglobulin precipitation method, caproic acid method, sad method, ion-exchange chromatography (DEAE or DE52) or using anti-Ig columns or albumin A, G or L columns affinity chromatography (it is all as described in more detail below that It a bit), can be from above-mentioned culture supernatants or ascites separation and antibody purification.

When building immunoglobulin molecules, variable region or part thereof can melt with one or more constant regions or part thereof It closes, connect or be otherwise connected, to generate any antibody as described herein.This can be in a number of ways known in the art It realizes, including but not limited to：Molecule clone technology, or direct molecule described in composite coding nucleic acid.

Bonding agent, antibody or its segment as " immunoreactive " expression used herein：They are residual to amino acid The sequence (" binding site " or " epitope ") of base is specific, and if there is cross reactivity with other peptide/albumen, The level that they are prepared to be applied to people's purposes is nontoxic.Term " in conjunction with " indicates in physiological conditions due to for example total Valence, electrostatic, hydrophobic and ion and/or interaction of hydrogen bond and binding directly between 2 molecules occurring, including interaction Such as salt bridge and water bridge and any other conventional combination.Term " preferentially combining " indicates, unrelated amino is combined with it Acid sequence is compared, and bonding agent is with the affinity combination binding site of bigger.With bonding agent to the affine of unrelated amino acid sequence Power is compared, affinity can be big at least 1 times, it is at least 2 times big, at least 3 times big, at least 4 times big, at least 5 times big, big at least 6 Times, it is at least 7 times big, at least 8 times big, at least 9 times big, 10 times, it is at least 20 times big, at least 30 times big, at least 40 times big, big at least 50 times, greatly at least 60 times, greatly at least 70 times, greatly at least 80 times, greatly at least 90 times, greatly at least 100 times or greatly at least 1000 times.Art Language " immunoreactive " and " preferentially combining " use interchangeably herein.

Terms used herein " affinity " indicate the equilibrium constant of the Reversible binding of 2 kinds of reagents, and are expressed as Kd.In conjunction with Albumen can be to the affinity (affinity of such as antibody to epitope) of ligand, for example, about 100 nanomoles (nM) are to about 0.1 NM, about 100 nM are to about 1 picomole (pM) or about 100 nM to about 1 femtomole (fM).Terms used herein " affinity " table Show, the compounds of two or more reagents is after dilution to the resistance of dissociation.Pass through such as enzyme linked immunosorbent assay (ELISA) (ELISA) or the methods of any other technology familiar to the person skilled in the art apparent affinity can, be measured.By such as Scatchard is analyzed or the methods of any other technology familiar to the person skilled in the art, can measure affinity.

" epitope " expression, antigen or other macromoleculars can form binding interactions with the variable region engagement groove of antibody Part.Such binding interactions can be shown as point with one or more amino acid residues of one or more CDR It is contacted between son.Antigen binding can be related to, for example, CDR3 or CDR3 pairs, or in some cases, V_HAnd V_LUp to all the 6 of chain The interaction of a CDR.Epitope can be linear peptide sequence (that is, " continuous "), or can be by discontinuous amino acid sequence (that is, " conformation " or " discontinuous ") is constituted.Antibody can identify one or more amino acid sequences；Therefore, epitope can be with Limit more than one unique amino acid sequence.Pass through peptide mapping well known to the skilled person and sequence analysis skill Art can measure the epitope identified by antibody.Binding interactions are shown as point with one or more amino acid residues of CDR It is contacted between son.TRC105 is a kind of mouse antibody, it be in 5,928,641,6,200,566,6,190,660 He of U.S. Patent number It is the identical amino acid sequence of mouse antibody of Y4-2F1 or SN6j described in 7,097,836.Identified in the past Y4-2F1 and SN6j identifies the epitope of (thus TRC105 is also identified).

Term is " specific " to indicate such situation：Wherein antibody is no longer shown to the table in addition to being identified containing antibody Any notable combination of molecule other than the antigen of position.The term is also applied for such situation：For example, antigen-binding domains It is specific to the defined epitope that many antigens carry, in this case, antibody can be in conjunction with the difference for carrying the epitope Antigen.Term " preferentially combining " or " specifically combining " indicate, compared with it combines unrelated amino acid sequence, antibody with The affinity combination epitope of bigger, and if the polypeptide for containing the epitope with other has cross reactivity, be at them The level for being applied to people's purposes and preparing is nontoxic.In one aspect, with antibody to the affine of unrelated amino acid sequence Power is compared, and such affinity is at least 1 times big, at least 2 times big, at least 3 times big, at least 4 times big, at least 5 times big, big at least 6 times, it is at least 7 times big, at least 8 times big, at least 9 times big, 10 times big, at least 20 times big, at least 30 times big, at least 40 times big, big At least 50 times, greatly at least 60 times, greatly at least 70 times, greatly at least 80 times, greatly at least 90 times, greatly at least 100 times or greatly at least 1000 Times.Term " immunoreactive ", " in conjunction with ", " preferentially combining " and " specifically combining " makes interchangeably herein With.Term " in conjunction with " indicate in physiological conditions due to for example covalently, electrostatic, hydrophobic and ion and/or interaction of hydrogen bond and Binding directly between 2 molecules occurred, including interaction such as salt bridge and water bridge and any other conventional combination side Formula.

When applied to polypeptide, " separation " (being interchangeably used with " substantially pure ") indicates polypeptide or part thereof, Itself because it source or operation and：(i) it is present in host cell, the expression product of the part as expression vector；Or (ii) albumen or other chemical parts other than those of being connected in nature with it are connected；Or (iii) in nature not In the presence of for example, the albumen of following chemical operation：At least one hydrophobic part is added or added to the albumen so that the egg It is white to be in the form being not present in nature." separation " in addition indicates such albumen,：(i) it is chemical synthesis；Or (ii) it expresses in host cell, and is purified from the albumen and contaminating protein of combination.As the term usually indicates Polypeptide：Natively adjoint other albumen and nucleic acid separate with it for it.In general, the polypeptide also with for purifying its Substance detaches, the substance such as antibody or gel-type vehicle (polyacrylamide).

Angiogenesis term

Terms used herein " Agiogenesis inhibition ", " inhibiting angiogenesis " or " anti-angiogenesis " include blood vessel Occur（vasculogenesis）Inhibition, and be intended to mean that realize new vessels formed degree, amount or rate reduction.It is real The reduction of the endothelial cell proliferation or degree of migration, amount or rate in now organizing is the specific example for inhibiting angiogenesis.

Term " composition of Agiogenesis inhibition " indicates such composition：It inhibits the mistake of angiogenesis-mediated Journey, such as endothelial cell migration, proliferation, pipe are formed, and subsequently result in the inhibition that new blood vessel is generated from existing blood vessel, and thereby shadow Ring the situation of angiogenesis-dependent.

Terms used herein " situation of angiogenesis-dependent " are intended to mean that such situation：Wherein angiogenesis or The process of angiogenesis is supported or increases pathological state, or advantageously influences normal physiology course.Therefore, wherein angiogenesis Support the treatment of the situation of the angiogenesis-dependent of pathological state that may lead to the alleviation of disease, and wherein angiogenesis is advantageous The treatment that ground influences the situation of the angiogenesis-dependent of normal physiology course may lead to the enhancings of such as normal processes.

Angiogenesis is to form new blood vessel from pre-existing capillary or post capillary venules.Angiogenesis source Since the formation for the new blood vessel that angioblast (they are precursors of endothelial cells) generates.Two processes lead to new blood vessel shape At, and be included in the meaning of situation of term angiogenesis-dependent.Terms used herein " angiogenesis " are intended to packet It includes, blood vessel re-forms, the blood vessel such as generated from angiogenesis and point from existing blood vessel, capillary and venule The blood vessel that branch and growth generate.Angiogenesis relates to, and induces ALK1 signal transductions and 1/5/8 phosphoric acid of related Smad Change and/or signal transduction.It it is known that CD105 is related to ALK1 signal transduction paths, thus be also included in the meaning of angiogenesis It is interior.

The CD105 for starting tumour is found that in people's kidney⁺Cell colony.CD105⁺Cell show before about The feature of tumor stem cell described in the cancer stem cell present in other tumor types.The CD105+ cells observed are shapes At clonal, expression stem cell markers, and lack differentiation marker, epithelium and endothelial cell class can be divided into vitro Type, and the tumour that can continuously transplant can be generated in vivo.Although the clone from expression interstitial marker, on the tumour is Skin cancer (as source tumour), and be characterized in that：CD105⁺The CD105 of the maintenance of tumorigenesis group and the differentiation of non-tumorigenic^- The presence of group.

" induction host immune response " indicates that the mitigation or reduction of the S or S of patient experience disease are specifically wrapped Include but be not limited to the extension of survival period.

Terms used herein " proliferative disorders " and " proliferative conditions " indicate, are characterized in that abnormal or undesirable proliferation Any pathological or non-pathological physiological status.Term " cell proliferative diseases " and " cell proliferative condition " indicate, special Sign is any pathological or non-pathological physiological status of abnormal or undesirable cell Proliferation, and including：It is special Sign is undesirable or unwanted cells proliferation or the situation of cell survival (for example, due to defective Apoptosis), It is characterized in that the situation of defective or abnormal or defective Apoptosis, and is characterized in that abnormal or undesirable Or the situation of unwanted cells survival.Term " differentiation illness " indicates, is characterized in that appointing for abnormal or defective differentiation What pathological or non-pathological physiological status.

It is obedient to the proliferation for the treatment of or breaks up illness and includes：It is characterized in that abnormal or undesirable cell number, cell life Long or cell survival benign and tumorous disease condition.Therefore such illness or situation may be constructed morbid state, And include cell, tissue or the organ of all types of cancerous growths or oncogenic process, transfer tissue or vicious transformation.

Including the cell of proliferation or differentiation illness can be gathered into cell mass, or disperse." non-physical knurl " indicates：It makes The tumor of blood system is formed, such as lymthoma, myeloma and leukaemia, or is that the tumor spread is formed in nature, because they are logical Often do not form solid block.The specific example of leukaemia includes：For example, acute and chronic lymphoblastic leukemia, pulpefaction are thin Born of the same parents' leukaemia and Huppert's disease.

Term " solid tumor " indicates that the tumor for being usually concentrated in together and being formed block is formed or shifted.Such illness includes swollen Tumor or cancer, they can influence substantially any cell or tissue type, for example, cancer, sarcoma, melanoma, transfer venereal disease Disease or hematopoetic tumor venereal disease disease.Metastatic tumo(u)r can be originated from a variety of primary tumor types, including but not limited to breast, lung, Thyroid gland, incidence, brain, lymph sample, gastrointestinal tract (mouth, esophagus, stomach, small intestine, colon, rectum), urogenital tract (uterus, ovum Nest, uterine neck, bladder, testis, penis, prostate), kidney, pancreas, liver, bone, muscle, skin etc..

Cancer indicates the malignant tumour of epithelium or endocrine tissue, and includes respiratory system carcinoma, gastrointestinal system cancer, uropoiesis life Grow system cancer, carcinoma of testis, breast cancer, prostate cancer, internal system cancer and melanoma.Illustratively cancer includes：From uterine neck, Those of lung, prostate, breast, incidence, colon, liver and ovary formation.The term also includes carcinosarcoma, for example, they are wrapped Include the malignant tumour being made of carcinous and sarcomatous tissues.Gland cancer includes that the cancer of glandular tissue or in which tumour form adenoid structure.

Cancerous tissue to be treated is, for example, the endothelial tissue of the CD105 of abnormal expression level or the cell of conversion.This The term " cell of conversion " that text uses indicates, has spontaneously been converted to the cell without limitation growth conditions, that is, they are Obtain the ability grown by unlimited number of division in culture.It, can be with all in terms of they grow control missings Such as tumorous, denaturation and/or proliferative term characterizes the cell of conversion.For the purposes of the present invention, term " the conversion phenotypes of malignant mammalian cells " and " conversion phenotype " are intended to, thin with mammalian cell The related any following phenotypic characteristics of dysuria with lower abdominal colicization：Immortalization, form or growth conversion and tumorigenicity are (by cell culture In extension growth, the growth in semisolid culturemedium or the cause tumour growth in immunoincompetent or homologous animal To detect).

Term " tumor-cell antigen " is defined herein as, the tumour cell of nothing to do with, normal cell or normal It is compared in body fluid, antigen is present in higher amount on tumour cell or in body fluid.By well known by persons skilled in the art Any number of experiment, can test antigen presence, and the experiment includes but not limited to the feminine gender and/or positive choosing using antibody It selects, such as ELISA experiments or pass through western blot at radiommunoassay.

Term " Apoptosis " or " programmed cell death " indicate such physiology course：In development and other normal lifes During object, undesirable or useless cell is eliminated by the process.Apoptosis is issued in normal physiological conditions Raw cell death pattern, cell are dead active participant'ss (" cell suicide ") of own.It is most commonly in following mistakes Cheng Zhong：Normal cell turnover and tissue homeostasis, embryo occur, the induction of immunological tolerance and maintenance, nervous system The Telatrophy of development and endocrine dependence.The cells show of experience Apoptosis goes out distinctive morphology and biochemical character. These features include：Chromatin agregation, nucleus and cytoplasm concentration, cytoplasm and nucleus separate the vesica that film forming combines Complete mitochondria and caryoplasm on (apoptotic body, they contain ribosomes), morphology.In vivo, these apoptotic bodies are quick Ground is identified and is swallowed by macrophage, dendritic cells or neighbouring epithelial cell.Due to removing this of apoptotic cell in vivo Effective mechanism will not cause inflammatory response.In vitro, apoptotic body and remaining cell fragment finally expand, and finally split Solution.The latter stage of the Cell death in vitro has been referred to as " secondary necrosis ".It by methods known to those skilled in the art, can be with Measure Apoptosis, the method such as exposure of DNA fragmentation, annexin V, the work of Caspase Change, the release etc. of cromoci.The cell of inducing death is herein referred to as " apoptotic cell ".

" cell death inducer " is defined herein as inducing cell apoptosis/programmed cell death, and includes, example Such as, anti-CD105 antibody, anti-VEGF antibodies, irradiation, chemotherapeutics or receptor bridging agent, wherein cell (for example, tumour cell or Endothelial cell) it is induced to undergo programmed cell death.Illustrative cell death inducer has been described in greater detail below.

It, can be with test cell apoptosis using the annexin V Apoptosis assay of standard：The culture in 6- orifice plates (NUNC) NIH:OVCAR-3 cells, irradiation, or handled 4-48 hours with antagonist (or combined with another anticarcinogen), washing is used in combination Annexin V-FITC (BD-Pharmingen) is dyed 1 hour.By flow cytometry (Becton-Dickinson, CellQuest cell) is analyzed, with propidium iodide counterstain, and is analyzed again in flow cytometry.

The method for preparing and expressing antibody

By means of genetic engineering, chimeric immunoglobulin has been had been built up.What is had previously described is most of chimeric immune The included V from mouse monoclonal antibody of globulin_HAnd V_LWith the C of human antibody_LAnd Fc.It can use next described herein Any isotype the areas Fc.It is as described herein it is chimeric can also include such standard：By the standard, light chain is modified A limited number of amino acid in the frame of variable region and/or weight chain variable chain, to increase the affinity of antibody.

With regard to for for ruling treatment by men, chimeric antibody usually to have several potential advantages better than mouse antibodies.Because of antibody Effect part be people, it is believed that it can preferably interact with the other parts of human immune system (for example, by complement according to The cytotoxicity (CDC) of property or the cytotoxicity (ADCC) of the cell of antibody dependent is relied more effectively to destroy target cell).Separately Outside, the constant region of chimeric antibody will not be identified as exotic by human immune system, therefore for the anti-of the antibody of such injection Body response should usually less than be directed to the antibody response of complete external mouse antibodies.Finally, it is known that mouse antibodies are recycled in people In half-life period be significantly shorter than half-life period of human antibody.It is assumed that chimeric antibody can have with naturally occurring human antibody more Similar half-life period, this allows to apply smaller and more low-frequency dosage.

It, can be extraly with residual in the CDR of other amino acid replacement antibody when it is expected the affinity of increased antibody Base.It is no more than 4 amino acid residues in general, changing in CDR, more generally, changes in CDR and be no more than 2 residues, still Except heavy chain CDR2, it at this moment can change up to 10 residues.By conventional method, it is all as those described herein (for example, Biacore), the variation of affinity can be measured.

Using routine techniques known in the art, antibody can be built and produced.In addition, can often be recombinated with mass production The antibody of preparation, especially when using high level expression carrier.

For veterinary applications, by using inhuman Fc, can synthesize for being administered to non-human (for example, primate is dynamic Object, ox, horse, pig etc.) antibody.

Using the recombinant technique at restriction endonuclease site, art-recognized technology can be used (such as Provided herein is with those of be incorporated to) nucleotide of modification encoding amino acid sequence.

In order to express, expression system is such system：It utilizes GS systems (Lonza), uses glutamine synthelase Gene alternatively marks.In brief, it using GS systems (Lonza), is alternatively marked using glutamine synthetase gene Note, by electroporation (250V), is transfected in Chinese hamster ovary celI.In the fetal calf serum containing 10% dialysis, (FCS contains 2 mM Glutamine) DMEM (Sigma) in, cultivate wild-type CHO cells.By electroporation, with the DNA of 300 μ g linearisations, transfection 6x10⁷Chinese hamster ovary celI.After electroporation, cell is resuspended in the DMEM containing glutamine, bed board is in 36x96- On orifice plate (50 holes μ l/), in 5% CO₂In at 37 DEG C be incubated.Next day, the selective medium that 150 holes μ l/ are added (do not have paddy The DMEM of glutamine).After about 3 weeks, using unrelated antibody as negative control, sieved by ELISA (see below)s Select bacterium colony.It will production>In all bacterium colonies amplification to 24- orifice plates of 20 μ g/ml, then expand into double T25 flasks.

In order to which high level produces, widely used mammalian expression systems are such systems：It is using by dehydrogenation leaf The gene magnification operation that hydrochlorate reductase-deficient (" dhfr- ") Chinese hamster ovary cell provides.The system is based on dehydrogenation Folate reductase "dhfr" gene, gene code DHFR enzymes, the conversion of the enzymatic dehydrofolic acid salt to tetrahydrofolate. In order to realize high yield, dhfr- is transfected with the expression vector containing functional DHFR genes together with the gene of encoding target albumen Chinese hamster ovary celI.In this case, target protein is recombinant antibodies heavy chain and/or light chain.

By increasing the amount of competitiveness DHFR inhibitor methotrexate (MTX) (MTX), recombinant cell passes through amplificationdhfrGene comes Generate resistance.Nominally, the amplification unit of use is far longer thandhfrThe size of gene, as a result coamplification antibody weight Chain.

When it is expected to mass produce albumen (such as antibody chain), the expression and stability of the cell being considered as. In long-term cultivation, recombinaant CHO cell group loses same in terms of their specific antibody productivity in amplification procedure Matter, even if they are originated from single Parental clones.

This application provides the polynucleotides (nucleic acid) for the separation for encoding antibody as described herein or part thereof, containing this The carrier of polynucleotides and for the host cell and expression system by this polynucleotides transcription and translation at polypeptide.

The application also provides the construct of plasmid, carrier, transcription or expression cassette form, and it includes at least one above-mentioned more Nucleotide.

The application also provides recombinant host cell, and it includes one or more above-mentioned constructs.It encodes described herein The nucleic acid of any antibody form the one side of the application, the production method of antibody also forms the one side of the application, institute The method of stating includes：Expression is realized from by the code nucleic acid in its source.By cultivating the weight containing the nucleic acid under proper condition Group host cell, can easily be expressed.After by Expression product, using any suitable technology, it can detach And/or antibody purification or part thereof, then it is suitably used.

It can be provided, detached and/or purified in the form of substantially pure or homogeneity from such as their natural surroundings Encoding particular antibodies (or part thereof) nucleic acid molecules and carrier containing nucleic acid molecules as described herein.The nucleic acid the case where Under, free or the nucleic acid or gene source that are substantially free of other than encoding the sequence of the polypeptide with required function. Nucleic acid sequence can include DNA or RNA, and can be wholly or partially synthetic.Purification process is well-known in the art.

System for being cloned in a variety of different host cells and expressing polypeptide is well-known.Suitable host Cell includes bacterium, mammalian cell, yeast and rhabdovirus system.It can be used for the food in one's mouth of expressing heterologous polypeptide in the art Newborn animal cell line includes but is not limited to：Chinese hamster ovary cell, HeLa cells, baby hamster kidney cell, NS0 mouse bone marrow cells Oncocyte and many other cells.

A variety of unicellular host cells can also be used for expression DNA sequence dna.These hosts include well-known eukaryon and original Core host, Escherichia coli, pseudomonas, bacillus, streptomyces, fungi such as in tissue culture are (such as Yeast) and zooblast (such as CHO, YB/20, NS0, SP2/0, Rl.l, B-W and L-M cell, African green monkey kidney cell (example Such as, COS 1, COS 7, BSC1, BSC40 and BMT10)), the strain of insect cell (for example, Sf9) and people's cell and plant cell System.

Expression of the antibody or part thereof in prokaryotic cell (such as Escherichia coli) is fully established in the art.It closes In summary, see, for example, Pl ü ckthun, A.Bio/Technology 9: 545-551 (1991)。

Expression in the eukaryocyte of culture is also that those skilled in the art can be used as producing antibody as described herein One selection, it is newest summary see, e.g. Raff, M.E. (1993)Curr. Opinion Biotech. 4: 573- 576;Trill J.J. et al. (1995)Curr. Opinion Biotech6:553-560, each piece in them is with it It is incorporated herein by reference.

Suitable carrier can be selected or be built, appropriate regulatory sequence, including promoter sequence, terminator sequence are contained Row, polyadenylation sequence, enhancer sequence, marker gene and other sequences appropriate.Carrier can be plasmid appropriate, Viral vectors, such as bacteriophage or phasmid.About other details, see, e.g., Molecular Cloning: a Laboratory Manual:Second edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known nucleic acid manipulation technologies and method, for example, the preparation of nucleic acid construct, mutagenesis, sequencing, DNA imported it is thin With gene expression and the analysis of albumen in born of the same parents, it is described in detail in：Short Protocols in Molecular Biology, Second edition, Ausubel et al. are compiled, John Wiley & Sons, and 1992.The method of Sambrook et al. and Ausubel et al. Disclosure is incorporated herein by reference with it, and is well-known in the art.

Thus, another aspect provides a kind of host cells, contain nucleic acid disclosed herein.It provides on the other hand A kind of method, the method includes：Such nucleic acid is imported in host cell.The importing may be used any available Technology.For eukaryocyte, suitable technology may include, for example, calcium phosphate transfection, deae dextran, electroporation, lipid Transfection that body mediates and use retrovirus or other viruses (for example, cowpox, or for insect cell, baculoviral) Transduction.For bacterial cell, suitable technology may include, for example, calcium chloride conversion, electroporation and turn using bacteriophage Dye.

After importing, the expression from nucleic acid can be caused or allow, such as by cultivating place under the conditions of gene expression Chief cell.

In one embodiment, by nucleic acid integration into the genome (such as chromosome) of host cell.According to standard Technology can promote to integrate by the sequence comprising promotion with the recombination of genome.As needed, Ig enhancings can be initialized, So that expression maximizes.

The application also provides a kind of method, the method includes：Above-mentioned construct is used in expression system, so as to Express above-mentioned antibody (or part thereof).

The application also relates to the nucleic acid of separation, the gene of such as recombinant DNA molecules or clone or its degeneracy variant, mutation Body, analog or its segment, they encode the antibody in conjunction with CD105.

In another embodiment, the gene of the recombinant DNA molecules or clone of antibody as described herein or part thereof Global DNA sequence can be operatively attached on expression control sequence, and the latter can import in appropriate host.The application is therefore Unicellular host is extended to, the host is by the clone gene of antibody or includes the V of encoding antibody_H、V_L、C_LAnd/or the DNA of Fc The recombinant DNA molecules of sequence convert.

Another is characterized in, the expression of DNA sequence dna disclosed herein.As known in the art, it can express as follows DNA sequence dna：They are operably coupled on the expression control sequence in appropriate expression vector, and using the expression vector come Convert unicellular host appropriate.

This be operatively connected of DNA sequence dna and expression control sequence includes (if not the portion of DNA sequence dna of course Point)：In the correct reading frame of DNA sequence dna upstream, initiation codon ATG is provided.

Can by separation and/or in the form of purifying (for example, more with required function free or substantially free of coding The polynucleotides in the source other than the polynucleotides of peptide) polynucleotides and carrier are provided.It is used herein " substantially pure " and Substantially free indicates, outside less than for example, about 20% or less exotic, about 10% or less exotic, about 5% or less Carry out the molten of object, about 4% or less exotic, about 3% or less exotic, about 2% or less exotic or about 1% or less exotic Liquid or suspension.

A variety of hosts/expression vector combination can be used for expressing the DNA sequence dna of the present invention.Useful expression vector, for example, It can be made of the section of chromosome, non-chromosome and synthesis DNA sequence dna.Suitable carrier includes but is not limited to： The derivative of SV40 and known bacterial plasmid, for example, escherichia coli plasmid col El, Pcr1, Pbr322, Pmb9 and they Derivative, plasmid such as RP4；Phage DNA, for example, numerous derivatives of phageλ, for example, NM989 and other bacteriophages DNA, for example, M13 and filamentous single stranded phage DNA；Yeast plasmid such as 2u plasmids or derivatives thereof；It can be used in eukaryocyte Carrier, such as can be used for the carrier in insect or mammalian cell；The carrier of combination from plasmid and phage DNA, Such as it has been modified to the plasmid using phage DNA or other expression control sequences；Deng.

A kind of recombinant host cell is also provided herein, it includes one or more polynucleotide constructs.Coding is herein The polynucleotides of the antibody of offer form the one side of the application, and the production method of antibody also forms the side of the application Face, the method includes：It is expressed from one or more polynucleotides.For example, being contained by cultivating under proper condition The recombinant host cell for stating polynucleotides may be implemented to express.Then any suitable technology is used, can be detached and/or pure Change antibody, and is suitably used.

Any in a variety of expression control sequences (control is operably connected to the sequence of the expression of the DNA sequence dna on it) Kind can be used in these carriers, to express DNA sequence dna.Such useful expression control sequence includes, for example, the morning of SV40 Phase or late promoter, CMV, cowpox, polyoma or adenovirus,lacSystem,trpSystem, TAC systems, TRC systems, LTR systems, The major operator and promoter region of phageλ, the control zone of fd coat protein, 3-phosphoglycerate kinases or other sugared ferment Promoter, the promoter of acid phosphatase (for example, Pho5), the promoter of yeast alpha mating factor and the known control for solving enzyme are former The various combination of the other sequences and they of the expression of the gene or their virus of core or eukaryocyte.

It should be appreciated that and not all carrier, expression control sequence and host equally rise expression DNA sequence dna function. Also and not all host equally works to identical expression system.But without excessive experiment, those skilled in the art Carrier, expression control sequence and host appropriate can be selected, to realize desired expression, without departing from scope of the present application. For example, when selecting carrier, it is necessary to host is considered, because the carrier must function in the host.It further accounts for The copy number of carrier, the ability for controlling the copy number and any other albumen (such as antibiotic mark by the vector encoded Will object) expression.Those of ordinary skill in the art can select carrier, expression control sequence and host appropriate, to realize the phase The expression of prestige, without departing from scope of the present application.For example, when selecting carrier, host is considered, because the carrier must be in institute It states in host and functions.It is also conceivable to the copy number of carrier, controlling the ability of the copy number and by the vector encoded Any other albumen (such as antibiotic markers) expression.

The application also provides the construct in plasmid described elsewhere herein, carrier, transcription or expression cassette form, they Including at least one above-mentioned polynucleotides.Suitable carrier can be selected or build, containing regulatory sequence appropriate, including Promoter sequence, terminator sequence, polyadenylation sequence, enhancer sequence, selected marker and other sequences appropriate.Carrier Can be plasmid appropriate, virus, such as bacteriophage appropriate, phasmid etc..About other details, see, e.g., Molecular Cloning: a Laboratory Manual:Second edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known nucleic acid manipulation technologies and scheme, such as nucleic acid construct Preparation, sequencing, imports DNA in cell and gene expression and the analysis of albumen mutagenesis, is described in detail in：Short Protocols in Molecular Biology, second edition, Ausubel et al. are compiled, John Wiley & Sons, 1992.The method and disclosure of Sambrook et al. and Ausubel et al. are incorporated herein by reference with it.

When selecting expression control sequence, many factors are usually considered.These factors include, for example, system is relatively strong The compatibility of degree, its controllability and it and the specific dna sequence or gene to be expressed, especially in potential secondary structure side Face.By the energy for considering such as compatibility of carrier of they and selection, their secretion feature, their correct folded protein Power and their fermentation need and by the DNA sequence encoding to be expressed product to the pure of the toxicity of host and expression product Change easiness, selects suitable unicellular host.

Another aspect provides a kind of host cells, contain one or more polynucleotides disclosed herein.It is another A aspect is provided imports such one or more polynucleotides the side in host cell by any available technology Method.For eukaryocyte, suitable technology may include, for example, calcium phosphate transfection, deae dextran, electroporation, liposome are situated between The transfection led and use retrovirus or other viral (for example, cowpoxs), or for insect cell, baculoviral turns It leads.For bacterial cell, suitable technology may include, for example, calcium chloride conversion, electroporation and the transfection using bacteriophage.

After importing, can cause or allow the expression of one or more polynucleotides, for example, by from a kind of or A variety of polynucleotides cultivate host cell under conditions of expressing one or more polypeptides.Derivable system can be used, and is led to It crosses addition activator and carrys out induced expression.

In one embodiment, polynucleotides can be integrated into the genome (such as chromosome) of host cell. It can promote to integrate by the sequence comprising promotion with the recombination of genome according to standard technique.In another embodiment In, the nucleic acid maintains on the episomal vector in host cell.

There is provided herein such method, the method includes：Above-mentioned construct is used in expression system, so as to table Up to particular polypeptide.

In view of these and other factors, those skilled in the art can build variety carrier/expression control sequence/host Combination, they express the DNA sequence dna in fermentation or extensive animal culture.

Other than clone, or as the replacement of clone, can recombinate/synthetically prepare encoding antibody or part thereof Polynucleotides.Polynucleotides can be designed to have codon appropriate.In general, if sequence will be used to express, it will Selection preferred codon for target host.It can be complete from the overlapping oligonucleotide assembly prepared by standard method Polynucleotides, and it is assembled into complete encoding sequence.See, e.g., Edge,Nature, 292:756 (1981)； Nambair Et al.,Science, 223:1299 (1984)；Jay et al.,J. Biol. Chem., 259:6311 (1984)。

It, can be simultaneously by a variety of methods well known by persons skilled in the art, including such as recombination method and chemical synthesis Mix encoding antibody (or part thereof) nucleic acid and selection amino acid position variation.

Anti- CD105 antibody

Endothelial factor (CD105) is expressed as 180 kDa homodimer transmembrane proteins on cell surface.Outer domains are with height Affinity (50 nM) combines TGF-β 1 and -3 isotypes, and the cross-film of CD105 and intracellular domain share 71% sequence with beta glycan Row similitude.People's CD105 genes are located on chromosome 9q34 (to be identified) using fluorescence in situ hybridization, and code area contains 14 Exon has characterized 2 different isotypes (L and S) of the CD105 with the ability in conjunction with TGF- β.L-CD105 by 633 amino acid residue compositions, wherein 47 amino acid residues, in cytoplasmic tail, this is different from S-CD105, Hou Zheyou 600 amino acid residue compositions, have 14 amino acid cytoplasmic tails.But L-CD105 is dominant forms.CD105 exists Composition ground phosphorylation in endothelial cell, mainly on serine and threonine residues, and the phosphorylation is due in the cell Form active TGF- β RII.The combination of TGF- β and CD105 lead to the downward of phosphorylation, are similarly to be pressed down with protein kinase C The effect that preparation is observed.People's CD105 amino acid sequences contain three peptide arginines-in the exposed region of extracellular domain Gly-Asp (RGD).RGD peptide is in ECM protein (such as fibronectin, vitronectin, von The Willebrand factors (vWF), type i collagen and fibrinogen) on the crucial identification structure that finds, and by cell surface integrin Albumen identifies.Integrin attachment has been directed to hemostasis, thrombosis, angiogenesis and inflammation, they are that wherein endothelium plays pass Key effect process (Duff et al.,FASEB J., 17:984-992 (2003))。

CD105 is a member of TGF-β receptor family, is expressed by the endothelial cell in being proliferated.Endothelial cell proliferation Need normal CD105 horizontal.CD105 expression can be increased by hypoxic (by hypoxia inducible factor-1-α (HIF-1- α) Production), and protect hypoxic cell from Apoptosis.Several functions of CD105 are related with TGF-β signal transduction.TGF-β passes through Heterodimer receptor signal conducts, and the receptor is made of serine kinase, receptor I (RI) and receptor II (RII).TGF-β And the combination of the outer domains of receptor, the RII kinase activities of meeting exposed cell matter, the activity meeting phosphorylation TGF-β RI, after Then person can interact with downstream signal transduction object (such as Smad albumen).CD105 forms the one of TGF-β receptor complex Part, but it can have an independent existence on cell surface.In many cell in vitro, CD105 inhibits TGF-β signal to pass It leads.

CD105 is also in relation with other growth factors, such as activator protein A and bone morphogenetic protein (BMP) -10, -9, -7 With -2.The combination of TGF-β or other growth factor ligands and CD105 needs at least the presence of receptor RII, and own cannot be tied Close ligand.The combination of CD105 and receptor will not change their affinity to ligand itself.After combination, the cell of CD105 Matter structural domain is by TGF-β RI and TGF-β RII phosphorylations；Then TGF-β RI (but TGF-β RII will not) kinases is from by bluk recombination Object dissociates.

The phosphorylation level of CD105 expression inhibiting TGF-β RII, but increase the phosphorylation level of TGF-β RI, cause to increase The phosphorylation of the Smad 2 added, but it is increased without the phosphorylation of Smad 3.Since Smad 2 can be with a variety of transcription factors, auxiliary work Agent and inhibitor interaction, the Smad 2 of phosphorylation can play the integration object of multi-signal, be turned with adjusting gene Record.Thus, CD105 adjusts TGF-β function (passing through the interaction with TGF-β RI and TGF-β RII), and modifies downstream Smad The phosphorylation of albumen.

CD105 plays a part of to adjust the signal transduction of a variety of kinases receptors compounds of TGF-β superfamily, including TGF-β Receptor (TGF-β R), Activin receptor-sample kinases (ALK) and Activin receptor.In the presence of no CD105, TGF-β by The activation of body leads to the phosphorylation of SMAD albumen (SMAD 2 and 3), this inhibits endothelial cell growth.But TGF-β is to CD105 Activation adjust SMAD protein phosphorylations (phosphorylation for including SMAD 1,5 and 8).Final result is that TGF-β receptor activation is internal The release of the growth inhibition effect of chrotoplast (referring to Fig. 2).It is not surprising that anti-CD105 antibody or antisense oligonucleotides Prevention to CD105 activation synergistically plays a part of to inhibit endothelial cell growth with TGF-β.

The length of CD105 promoters is 2.6 kb, but does not contain TATA or CAAT transcription initiation boxes.But it has 2 A region rich in GC：The consensus motif and TGF-β response element of Sp1, ets, GATA, AP-2, NGF- β and Mad.Although In this way, CD105 has relatively limited cell distribution.Basal transcription level seem to need the sites ets (at position -68) and The sites Sp1, but express relatively limited (for example, being limited to endothelial cell) seem to be related to multiple regulatory regions, specifically, one At -1294 to -932, another is very close to transcription initiation site.CD105 is raised by TGF-β, this have shown that needs- The sites Sp1 at 37 to -29 places, further relate to the sites SBE of one or more upstreams abutted, the site combine Smads 3 and/ Or 4 (they are activated by TGF-β signal transduction).Hypoxemia is the common trait of ischemic tissue and tumour, and is vascular endothelial cell (EC) active stimulus of the CD105 gene expressions in.With the such effect of combination enhancing of TGF-β 1.The CD105 of up-regulation can To play self-protection effect in the EC under hypoxia stress.

Blood vessel EC is the main source of CD105.Other cell types (including it is vascular smooth muscle cells, fibroblast, huge Phagocyte, the leukaemia cell of preceding-B and myelomonocyte source and erythroid precursors) it is expressed in lower degree CD105。

CD105 is related to angiogenesis.Anti-sense experiment has confirmed, inhibits CD105 tables in HUVEC in combination with TGF-β 1 It reaches, leads to significantly inhibiting for extracorporeal blood vessel generation, this shows that CD105 is the Angiogensis component in endothelial cell.CD105 exists The mouse that other evidences of important function in angiogenesis are knocked out from CD105.There is no the mouse of CD105 to show a variety of blood Pipe and heart defect, cause in embryo's Deaths.The several vascular lesions instruction observed in the mouse of not CD105, Formation of the ripe blood vessel outside embryo in vascular system needs CD105, this further demonstrates that CD105 is in angiogenesis Directly act on.

CD105 (being especially also referred to as endothelial factor or edg-1) is I type homodimer membrane glycoproteins, is existed with high level It is expressed in vascular endothelial cell in proliferation.Thus, CD105 is mainly the proliferation for the endothelial cell for undergoing active angiogenesis Relevant marker.But there may be limited CD105 expression for the blood vessel endothelium of normal structure.Known people CD105 specificity Ground bind to transforming growth factor-β (TGF-β), CD105 amino acid sequences and the β-glycan (type of TGF-β receptor of derivation Type) there is strong homology.

In the method for the reduction tumor vascular system based on antibody, CD105 is targeted, because CD105 is in endothelium With the relevant antigen of proliferation on leukaemia cell.Its expression in the relevant blood vessel endothelium of tumour is raised, and CD105 is Necessary to angiogenesis.Angiogenesis includes the formation of new capillary, this leads to new vessels formation and existing vascular The maintenance of system.This is a complicated process, which includes a series of successive steps, including the blood that endothelial cell mediates The degradation of pipe basement membrane and interstitial matrix, the migration of endothelial cell, the proliferation of endothelial cell and endothelial cell capillary loops It is formed.

CD105 can reside in：On the cell for constituting and supporting existing vascular system, and promote neovasculature On the cell for growing and becoming a part for neovasculature.These antibody can be in conjunction with CD105, and thereby inhibits blood vessel life At, inhibit the maintenance of existing vascular system or existing vascular system and/or inhibit thin vessels expansion.In addition to they are for purifying Other than the purposes of CD105, these antibody can be used for purifying, detect and diagnose purpose and therapeutic purposes.Antibody provided herein Can be used for being formulated for treating the drug of a variety of situations and disease, the method and detection method of the treatment situation and disease or Diagnostic method.Angiogenesis used herein includes growth and/or development (also referred to as new vessels are formed), the small blood of new blood vessel The expanding of pipe, excessive or extended angiogenic growth and existing vascular system maintenance.Angiogenesis situation and disease indicate with Associated angiogenesis is caused by angiogenesis or with those of angiogenesis disease and situation.Such disease it is unrestricted Property example includes, for example, various forms of cancers (primary tumor and transfer).The mouse Dan Ke for CD105 is produced Grand antibody (mAb), they adjust CD105 activity, and thereby inhibit angiogenesis and/or inhibit the vasodilation of thin vessels.This A little mouse antibody descriptions are each in them in United States Patent (USP) 5,928,641,6,200,566,6,190,660 and 7,097,836 A piece is hereby incorporated by reference in its entirety.In addition, it was verified that the ex vivo and internal validity of many such antibody；Knot The monoclonal antibody of conjunction CD105 is interesting as the compound of CD105 is adjusted.But the therapeutic of mouse antibody answers With being infeasible, because the application of mouse antibody has many limitations, including such as people's anti-mouse antibody (HAMA) form is exempted from Epidemic focus.

Several anti-CD105 antibody have been described, especially anti-CD105 monoclonal antibodies (" mAb ").MAb SN6 Be with the glycoprotein mixture of the cell membrane of human leukemia cell be immunized antibody that mouse generated (Haruta and Seon, 1986, Proc. Natl. Acad. Sci. 83:7898-7902).SN6 is a kind of mouse mAb of identification people CD105.MAb 44G4 be the full cell suspending liquid of-B leukaemia cells before employment be immunized antibody that mouse generated (Gougos and Letarte, 1988, J. Immunol. 141:1925-1933; 1990, J. Biol. Chem. 265:8361-8364).44G4 is also A kind of mouse mAb of identification people CD105.MAb MJ7/18 be generated with the mouse skin immune rat of inflammation antibody (Ge and Butcher, 1994, ibid).MJ7/18 is also a kind of mAb of identification mouse CD105.MAb Tec-11 are in human umbilical vein Chrotoplast be immunized mouse generated antibody (Burrows et al., 1995,Clin. Cancer Res. 1:1623-1634)。 Tec-11 is a kind of reactive mouse mAb for having and being limited to people CD105.This document describes the chimeric antibody for combining CD105, tables Reveal the immunogenicity of reduction, while maintaining and/or improving their specificity.In addition, related with mouse antibody in order to solve Problem shows to reduce immune this document describes CD105 is combined and the chimeric antibody of reduction and/or inhibition angiogenesis Originality, while maintaining and/or improving their specificity.These anti-CD105 antibody can be used for the various situations of diagnosing and treating And disease, and for purifying and detecting CD105.It is represented for the antibody of CD105 for treating a variety of diseases and situation (they Be related to angiogenesis, by angiogenesis influenced or effect) therapy development a key areas.

There is provided herein its antibody for combining CD105.Their antibody (or antigen-binding fragment) is also provided, it is described Antibody combination CD105, and inhibit (partially or even wholly) or control/treatment (partially or even wholly) angiogenesis/new life Vascularization, thin vessels expansion inhibit cell Proliferation or inhibit tumour growth.Similarly, in the meaning for inhibiting or combining CD105 It is interior, also include inhibiting CD105 functions (for example, signal transduction, combination, activation etc.).In another embodiment, antibody passes through Inhibit angiogenesis in conjunction with CD105.The application also provides the cell line that can be used for producing antibody, described thin for producing Method, the method for expressing antibody and purifying them of born of the same parents system.

It will recognize, using conventional method (including, but are not limited to ELISA), using provided herein or this field The experiment known can test the energy of the combination CD105 of the antibody for specifically combining CD105 generated using methods described herein Power.Using conventional method, including but not limited to Biacore or surface plasma body resonant vibration, antibody described herein can also be measured Affinity.

There is provided herein the antibody for combining CD105.It has been also provided herein in conjunction with CD105 and has inhibited the antibody of angiogenesis.

There is provided herein a kind of antibody, it includes：With SEQ ID NO:The light chain variable of amino acid sequence described in 1 Area has SEQ ID NO:The constant region of light chain of amino acid sequence described in 2 has SEQ ID NO:Amino acid described in 3 The heavy chain variable region of sequence and have SEQ ID NO:γ 1 (γ 1) constant region (Fc) of amino acid sequence described in 4.SEQ ID NO:Described in 3；With with SEQ ID NO:The constant region (Fc) of amino acid sequence described in 4.

In another non-limiting embodiment, the immune anti-CD105 antibody that goes of the humanization of separation can include With such as SEQ ID NO:The heavy chain variable region of amino acid sequence shown in 11 and with such as SEQ ID NO:Amino acid sequence shown in 12 The light chain variable region of row.Humanization-goes other non-limiting examples of immune heavy chain to include, but are not limited to SEQ ID NO:13, 14,15 and 16.Humanization-goes other non-limiting examples of immune light chain to include, but are not limited to SEQ ID NO:17,18, 19,20,21,22 and 23.The sequence is provided below in embodiment 17.

On the other hand, this application provides a kind of antibody, can under certain condition with it is as described herein anti- CD105 antibody competitions, wherein in ELISA experiments, it is described anti-by having for the competition blocking at least 5% with such antibody The V of body_HAnd V_LThe antibody of sequence is combined with CD105.

There is provided herein neutralizing antibodies, in conjunction with CD105, and adjust the activity of CD105.Neutralizing antibody can for example pass through Inhibit angiogenesis in conjunction with CD105.

The detection or diagnostic uses that antibody described herein can be used for being described in greater detail below.Antibody described herein It can be used for combining CD105, this can inhibit angiogenesis as described herein again.

Antibody as described herein can additionally comprise the treatment part for treatment use.

Antibody as described herein is also used as immunoconjugates.As used herein, in order to illustrate book and claim The purpose of book, immunoconjugates refer to by anti-CD105 antibody according to the present invention or its segment and at least one treatment marker structure At conjugate.Treatment marker includes anti-tumor agent comprising salmosin and angiogenesis inhibitors.Such anti-tumor agent comprising salmosin be this field Know, including but not limited to：Toxin, drug, enzyme, cell factor, radionuclide, pdt agent and angiogenesis inhibitors. Toxin includes but is not limited to：Ricin A chains, saltant type Pseudomonas exotoxin, diphtheria toxoid, broneomycin, rich peace are mould Plain (boamycin), saporin, gelonin and pokeweed antiviral protein.Drug include daunorubicin, methotrexate (MTX) and Calicheamicin.Radionuclide includes radioactive metal.Cell factor includes but is not limited to：Transforming growth factor (TGF)-β, Interleukin, interferon and tumor necrosis factor.Pdt agent includes but is not limited to：The derivative of porphyrin and they.Additional Treatment marker is known in the art, and is also considered herein.For making anti-CD105 mAb or its segment and at least one The compound method of antitumor agent be it is well known to the skilled person (that is, Ghetie et al., 1994,Pharmacol. Ther. 63:The antibody conjugates that 209-34 is summarized).Such method can utilize several workable for being coupled or connecting Connect one of the miscellaneous bifunctional reagent of molecule.Additional radionuclide (is such as treated and is diagnosed for connection molecule with additional Label) method be described further herein together.

Using technology for various purposes known in the art, such as, for example, by adding polyethylene glycol (PEG), it can To modify antibody.PEG modifications (Pegylation) can cause following one or more：Improved circulation time improves Solubility, improve to the resistance of proteolysis, the antigenicity of reduction and immunogenicity, improved bioavilability, reduce Toxicity, improved stability and be easier prepare (about summary, referring to Francis et al., International Journal of Hematology 68:1-18, 1998)。

The parts Fc that antibody can be modified, to increase the circulating half-life when being administered to patient in blood.Use this The usual manner in field, such as, for example, in U.S. Patent number 7,217,798 (it is incorporated herein by reference with it) The mode can measure modification.

The other methods for improving the half-life period of the fusion protein based on antibody in the circulating cycle are also known, such as, for example, Method described in U.S. Patent number 7,091,321 and 6,737,056 (each is incorporated herein by reference).In addition, It can produce or express antibody so that they do not contain the fucose on the sugar chain of their complicated N- glucosides-connection.? Know the effector function for increasing antibody and antigen-binding fragment from the sugar chain removal fucose of complicated N- glucosides-connection, including But it is not limited to：The cytotoxicity (CDC) of the cell-mediated cytotoxicity (ADCC) and complement-dependent of antibody dependent.It is similar Ground, can by combine CD105 antibody their ends C- end be connected to from any antibody isotype (for example, IgG, IgA, IgE, IgD and IgM) and any isotype subclass (especially IgG1, IgG2b, IgG2a, IgG3 and IgG4) all or On partial immunity immunoglobulin heavy chain.

Furthermore it is also possible to modify antibody as described herein so that they can pass through blood-brain barrier.Antibody as described herein This modification allow treat cerebral disease, such as glioblastoma multiforme (GBM).In U.S. Patent Publication Being described in 20070082380 (it is incorporated herein by reference with it) allows albumen (such as antibody) to pass through blood-brain barrier Illustrative modification.

It has been shown that the glycosylation of immunoglobulin is to their effector function, structural stability and from antibody producing Cell secretion rate have significantly affect (Leatherbarrow et al.,Mol. Immunol. 22:407 (1985)).It is negative The carbohydrate group for blaming these properties is generally connected in the constant area (C) of antibody.For example, the complement activation of IgG relies on The entire ability of the cytolytic classical pathway of property, needs IgG in C_HThe glycosylation at asparagine 297 in 2 structural domains (Tao and Morrison,J. Immunol. 143:2595 (1989)).IgM is in C_HAt asparagine 402 in 3 structural domains Glycosylation, be antibody it is appropriate assembly and cell lysis activity necessary to (Muraoka and Shulman,J. Immunol. 142:695 (1989)).In the C of IgA antibody_H1 and C_HThe glycosylation site at position 162 and 419 in 3 structural domains is gone Remove, cause intracellular degradation and at least 90% secretion inhibit (Taylor and Wall,Mol. Cell. Biol. 8:4197 (1988)).Furthermore it is possible to produce or express antibody so that they do not contain the sugar chain in their complicated N- glucosides-connection On fucose.The known effect for increasing antibody and antigen-binding fragment from the sugar chain removal fucose of complicated N- glucosides-connection Object function is answered, including but not limited to：The cell of cell-mediated cytotoxicity (ADCC) and complement-dependent of antibody dependent Toxicity (CDC).Using molecule clone technology known in the art, by multiple systems, these can be produced and " remove fucosylation " antibody, the technology includes but not limited to transgenic animals, genetically modified plants or cell line, their hereditarily engineerings Change so that they no longer contain for including enzyme and biochemistry necessary to fucose in the sugar chain of complicated N- glucosides-connection Approach (animal, plant or cell that also referred to as fucosyltransferase knocks out).Fucosyltransferase can be engineered to strike The non-limiting embodiment of the cell of the cell removed includes：Chinese hamster ovary celI, SP2/0 cells, NS0 cells and YB2/0 cells.

It has also been observed that glycosylation of the immunoglobulin in variable area (V).Sox and Hood reports, about 20% people are anti- Body be glycosylated in the areas V (Proc. Natl. Acad. Sci. USA66:975 (1970)).Think the glycosyl in V structure domain Change accidental appearance of the Glycosylation signals Asn-Xaa-Ser/Thr from N- connections in V region sequences, and this field is not yet thought It works in immunoglobulin function.

Glycosylation at variable domains Framework residues can change the binding interactions of antibody and antigen.The present invention Including such standard：By the standard, select the frame of immunoglobulin chain or a limited number of amino acid in CDR into Row mutation (for example, displacement, deletion or addition for passing through residue), to increase the affinity of antibody.

In general, being imported in the areas V frame by the way that one or more to be mutated, usually in the region of neighbouring one or more CDR In and/or in one or more framework regions, the affinity in conjunction with antigen can be adjusted.In general, such mutation is related to guarding The importing of amino acid replacement, the displacement destroys or establishes glycosylation site sequence, but has substantially no effect on the parent of polypeptide Hydrophobic (hydropathic) structural property.In general, avoiding importing the mutation of proline residue.In U.S. Patent number 6,350,861 The glycosylation of antibody is further described in (it is incorporated herein by reference about glycosylation).

It can be formulated for short tem delivery or the antibody of extended (long-term) delivering.

The CD105 that antibody in conjunction with CD105 can be used for purifying CD105 and/or detect in sample or patient is horizontal, with The disease related with CD105 or illness that detection or diagnosis are described in greater detail below.

Binding affinity, affinity and the neutralization of the antibody of the combination CD105 generated using such method can be tested It is one or more in ability.Useful antibody can be used for being administered to patient, with prevention, inhibition, control or treatment and blood vessel Generate related situation, disease or illness.

It can evaluate one or more in binding affinity, association rate, dissociation rate and the affinity of antibody.One A aspect can evaluate the active ability of the neutralization CD105 or VEGF of antibody.Using art-recognized experiment, including but It is not limited to：Enzyme linked immunosorbent assay (ELISA) (ELISA), Scatchard analyses, BIACORE analyses (surface plasma body resonant vibration) etc. And those of ordinary skill in the art it is usually used and it is known other experiment, can measure binding affinity, association rate, Dissociation rate and affinity.

Using such as enzyme linked immunosorbent assay (ELISA) (ELISA), competitive combination test, ELISPOT experiments or this field Any other the useful experiment known can measure the combination of antibody and CD105 and/or measure the inhibition blood vessel life of such as antibody At ability.These experiments are that those of ordinary skill in the art are usually used and well-known.

In one non-limiting embodiment, ELISA experiments can be used for measuring the knot of the specific antibodies in conjunction with CD105 Conjunction ability.

The experiments such as ELISA can be used for identifying shows the increased spy to CD105 compared with its other antibody Anisotropic antibody.The experiments such as ELISA can be used for identifying its such antibody：The antibody is combined across one or more The epitope of polypeptide and epitope across one or more CD105 or VEGF.By running parallel ELISA, specificity can be carried out Experiment, in the ELISA, the simultaneously one or more epitopes of the combination of screening experiment antibody in separated test chamber The ability of (on the different plant species polypeptide containing CD105 epitopes), to identify that it combines the antibody of CD105.People in the art Another technology for measuring apparent binding affinity known to member is Applications of surface plasmon resonance (in 2000 systems of BIACORE Analyzed on system) (Liljeblad, et al.,Glyco. J. 2000, 17:323-329)。Heeley, R. P., Endocr. Res. 2002, 28:217-229 describes canonical measure and traditional combination experiment.

Can also measure the treatment of antibody and the various disease of associated angiogenesis and the situation of specific binding CD105 with And the ability of various forms of cancers (for example, primary tumor, recurrent tumor and transfer).It is well known by persons skilled in the art Any suitable experiment can be used for monitoring this effect.This document describes several such technologies.In an example, it tests The ability of the combination CD105 of antibody described herein.In another example, it by surface plasma body resonant vibration (SPR), measures The affinity costant of antibody described herein.In another example, work of the antibody described herein to Agiogenesis inhibition is measured With.

Preparation

Other than active constituent (anti-CD105 antibody or its antibody fragment), preparation provided herein may include：Pharmaceutically Acceptable excipient, carrier, buffer, stabilizer, surfactant or other materials well known to the skilled person Material.Such material should be nontoxic, and the effect of will not interfere active constituent.The definite property of carrier or other materials will Depending on administration method.

There is provided herein the novel formulation of anti-CD105 antibody, the precharging injection syringe containing the preparation and such preparations It can be used for treating the purposes of angiogenesis associated disease.This application provides can be used for the application of intravenous or intraocular, for example, treatment With the preparation of CD105 relevant cancer and ophthalmic conditions.The knot of antibody or its antigen-binding fragment and CD105 as described herein Conjunction, affinity, affinity and activity (can include, but are not limited to embodiment above and below using art recognized methods Described in measuring method) evaluated.

In one aspect, the preparation is stable after preparation, can be tested according to conventional methods.Including albumen Preparation safe handling and application represent notable challenge to drug formulator.Albumen, which has, is presented stability problem Unique chemically and physically characteristic：For albumen, there are various degradation pathways, imply chemically and physically unstability.Chemistry is not Stability includes that deamination, aggregation, peptide backbone cut the oxidation cut with methionine residues.Physical instability includes many existing As, including, for example, polymerization.

" stabilization " preparation is that albumen therein substantially retains its physical stability and/or chemical stability after storage And/or the preparation of bioactivity.Various analytical technologies for measuring protein stability can be used and summarize in example in the art Such as, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A.Adv. Drug Delivery Rev. 10: 29-90 (1993).Stability can measure the period selected by selected constant temperature.Preferably, the preparation is in room Warm (about 30 DEG C) or lasting at least one moon is stable at 40 DEG C, and/or continues at least 1 year or at least 2 years to be at about 2-8 DEG C Stable.In addition, the preparation can be stable after the freezing (extremely, for example, -80 DEG C) and thawing of preparation.

If albumen by UV light scattering or passes through size exclusion after the visual inspection of color and/or clarity or such as Chromatography (SEC), which measures, shows the sign without aggregation, precipitation and/or denaturation, then albumen " it is steady to retain its physics in pharmaceutical preparation It is qualitative ".Method for measuring such measured value is known in the art, and is described in greater detail below.

If making the albumen be considered still retaining its biology as defined below in the chemical stability of given time Activity, then albumen " retain its chemical stability " in the formulation.Chemical stability can be changed by the chemistry of detection and Quantitative Western The form of change is evaluated.Chemical change can relate to size modification (being cut for example, cutting), can use such as size exclusion color Spectrum, SDS-PAGE and/or substance assistant laser desorpted ionized/flight time mass spectrum (MALDI/TOF MS) are evaluated.Other classes The chemical modification of type includes that charge changes (for example, occurring as the result of deamidation), can be for example, by ion exchange color Spectrum or Capillary isoelectric focusing (cIEF) are assessed.

If as example antigen binding assay measures, bioactivity of the antibody in given time is, for example, preparing In about the 50 to 150% of the biological activity shown when preparation (in evaluated error), then antibody " retains its life in the formulation Object activity ".For other " bioactivity " measuring methods of antibody, details are as follows.

It is such as measured by size exclusion chromatography method (SEC) for the preparation is with the stability of time, at least 95% anti-CD105 antibody or its antigen-binding fragment can be deposited after being stored at about 2 to 8 DEG C at least about 12 months as monomer ?.In addition, the art-recognized method for evaluating stability is also considered herein, including but not limited to described in embodiment Those.

In addition, stored at about 2-8 DEG C after at least about 12 months can be with for the anti-CD105 antibody or its antigen-binding fragment Display about 50 to 150% combines (passing through CD105 ELISA binding assays).

After being stored at least about 12 months at 2 to 8 DEG C, the average isoelectric point (pI) of anti-CD105 antibody can be about 8.8 to about 9.2, as by, for example, measured by the electricity focusing such as Capillary Electrophoresis.

After the freezing of the preparation and thaw cycle, as measured by SEC, the preparation can contain as single At least 95% anti-CD105 antibody existing for body.Alternatively, or additionally, when being subjected to agitation stress, preparation can contain As existing for monomer at least 95% anti-CD105 antibody.

Anti- CD105 antibody or its antigen-binding fragment can include that its CDR can be in conjunction with any sequence of CD105.One In a non-limiting embodiments, the anti-CD105 antibody is TRC105, and it includes with such as SEQ ID NO:Amino shown in 1 Light chain variable region (the V of acid sequence_L)；With such as SEQ ID NO:Constant region of light chain (the C of amino acid sequence shown in 2_L)；With such as SEQ ID NO:Heavy chain variable region (the V of amino acid sequence shown in 3_H)；With with such as SEQ ID NO:Amino acid sequence shown in 4 Constant region (Fc).

The preparation contains about 1 mg/ml to the anti-CD105 antibody of about 150 mg/ml or its antigen-binding fragment, or Any of which value, including but not limited to, about 2 mg/ml, about 5 mg/ml, about 7.5 mg/ml, about 10 mg/ml, 20 mg/ml, About 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, About 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, About 95 mg/ml, about 100 mg/ml, about 105 mg/ml, about 110 mg/ml, about 115 mg/ml, about 120 mg/ml, about 125 Mg/ml, about 130 mg/ml, about 135 mg/ml, about 140 mg/ml, about 145 mg/ml, about 150 mg/ml or more or its Between any integer.When being related to antibody concentration, terms used herein " about " indicate ± the 2% of institute's indicating value.

In one embodiment, the preparation includes the anti-CD105 antibody or antigen-binding fragment of about 25 mg/ml.? In another embodiment, the preparation includes the anti-CD105 antibody or antigen-binding fragment of about 50 mg/ml.Again another In embodiment, the preparation includes the anti-CD105 antibody or antigen-binding fragment of about 100 mg/ml.

Preparation as described herein can contain buffer, such as, for example, histidine, acetate, citrate or phosphoric acid Salt.May include buffers of about 5 mM to the amount of about 100 mM.In one embodiment, the preparation include about 5 mM, About 7.5 mM, about 10 mM, about 12.5 mM, about 15 mM, about 17.5 mM, 20 mM, about 22.5 mM, about 25 mM, about 30 MM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about Histidine, acetate, the citrate of 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM or in which any integer Or phosphate.When being related to buffer concentration, terms used herein " about " indicate ± the 2% of institute's indicating value.

Preparation can be used for becoming known for any kind of application of antibody, include, but are not limited in vitreum and vein Interior application.

In one embodiment, preparation provided herein is isotonic.Representative isotonic preparation includes, but are not limited to It is those of about 250 to about 350 millisomoles.In another embodiment, preparation provided herein is hypertonic.It is representative Hypertonic preparation includes, but are not limited to be those of about 351 to about 1000 millisomoles.

Polyalcohol can be added to preparation as described herein with the amount of at most about 1 M.For example, polyalcohol can include about 50 mM, about 75 mM, about 100 mM, about 150 mM, about 200 mM, about 225 mM, about 240 mM, about 250 mM, about 300 mM, About 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 The polyalcohol of the amount of mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM, about 1 M or in which any integer.At one In embodiment, preparation provided herein contains the polyalcohol of the amount less than 300 mM, and the preparation is made into isotonic, Wherein salinity is about 100 mM to about 175 mM.For example, the preparation containing the polyalcohol less than 300 mM amounts is made into isotonic , wherein salinity is about 130 mM.When being related to polyhydric alcohol concentration, terms used herein " about " expression institute indicating value ± 2%.In one aspect, the polyalcohol being ready to use in preparation provided herein can be sugar, such as, for example, nonreducing sugar.It is non- The representative example of reducing sugar includes, but are not limited to trehalose and sucrose.For example, the preparation can include about 200 mM extremely About 300 mM trehaloses or sucrose.In one embodiment, preparation can include about 240 mM trehaloses or sucrose.Alternatively, institute State the D-sorbite that sugar can be the amount (concentration) of about 200 mM to about 300 mM.In one embodiment, preparation may include About 240 mM D-sorbites.

Phos:Phosphate buffered saline (PBS)； His:Histidine。

Preparation provided herein can be with the pH of about 4.0 to about 7.5.In one embodiment, preparation provided herein It can be with about 4.5, about 5.0, about 5.5, about 6.0, about 6.5 or about 7.0 pH.Terms used herein " about ", when being related to pH, Indicate pH ± 0.05,0.1 or 0.2.

It will be understood by those skilled in the art that can prepare the preparation comprising antibody or antigen-binding fragment as follows (passes through this Method identification described in text) for storing：Albumen of the mixing with desired purity level and optional in form of an aqueous solutions Physiologically acceptable carrier, excipient and/or stabilizer (Remington's Pharmaceutical Sciences, the 16th Version, Osol, A. compile (1980)).

Acceptable carrier is the patient of application physiologically acceptable, and retain therewith/apply wherein The therapeutic properties of compound.Acceptable carrier and their preparation are, and are generally described in, for example, Remington ' (the 18th edition, A. Gennaro are compiled pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1990).A kind of illustrative carrier is physiological saline.Phrase " pharmaceutically acceptable carrier " used herein indicates acceptable Material, composition or medium, such as liquid or solid filler, diluent, excipient and/or solvent, they are related to leading Compound is inscribed to carry or transported to another organ or body part from the site of administration of an organ or body part.Each is carried Body is acceptable, and is meant that, compatible with the other compositions of preparation, and the subject applied to it is harmless.It is acceptable Carrier will not change the specific activity of motif compound.

In one aspect, there is provided herein pharmaceutically acceptable or physiologically acceptable compositions, including with application Compatible solvent (aqueous or non-aqueous), solution, emulsion, decentralized medium, coating agent, isotonic agent and sorbefacient or suction Receive delayed-action activator.Therefore, composition or preparation indicate, are suitble to the treatment in subject and/or the composition of diagnostic application.Combination Object and preparation include a certain amount of compound as described herein and pharmaceutically or physiologically acceptable carrier.

Composition can be configured to compatible with particular route of administration (that is, whole body or local).Thus, composition packet Include the carrier, diluent or excipient for fitting through different approaches to apply.

Composition can be applied, for example, by injection, including but not limited to：Subcutaneous, intravitreous, intradermal, Intravenous, endarterial, in peritonaeum or intramuscular injection.Can include isotonic agent in the composition, for example, carbohydrate, Polyalcohol (such as mannitol, D-sorbite) and sodium chloride.It is used as former state after obtained solution being packed, or freeze-drying；Institute The product for stating freeze-drying can be later combined with sterile solution before administration.For being injected intravenously or involving at position Injection, active constituent can be the form of parenterally acceptable aqueous solution, and the aqueous solution is pyrogen-free, and with conjunction Suitable pH, isotonic degree and stability.The related technical personnel of this field can expertly prepare suitable solution, wherein use example Such as isotonic medium, such as sodium chloride injection, ringer's injection, lactic acid salinization ringer's injection.As needed, it can wrap Include preservative, stabilizer, buffer, antioxidant and/or other additives.Aseptic injectable solution can be prepared as follows：It need to In the active constituent to be measured incorporation appropriate solvent, the solvent contains one of above-named ingredient or combination (as needed), Then filtration sterilization.

In one embodiment, preparation as described herein does not include surfactant.In another embodiment, originally Preparation described in text optionally includes surfactant, such as, for example, polysorbate 20 or 80, TWEEN^®、PLURONIC^®F68 or polyethylene glycol (PEG).

When considering composition in drug or any method provided herein, consider, the composition can be It is substantially pyrogen-free so that the composition will not cause inflammatory reaction or unsafe abnormal anti-when being administered to people patient It answers.It tests the pyrogen of composition and prepares substantially pyrogen-free composition, be that those of ordinary skill in the art are readily comprehensible, And it can be realized using commercially available packaging.

Phrase is " pharmaceutically acceptable " to indicate such molecular entity and composition：When being administered to subject, it is Pharmaceutical formulation, and usually not will produce allergic or similar adverse reaction, stomach upset, dizziness etc..

When referring to therapeutic combination, term " unit dose " expression is suitable as the unit dose of subject physically The unit of separation, each unit, which contains, to be computed with the active material of the predetermined amount that generates desired therapeutic effect and needs Diluent (that is, carrier or medium).

Composition can be applied in a manner of compatible with dosage particles and with therapeutically effective amount.Amount to be administered depends on waiting for The subject for the treatment of, the immune system of subject utilize the degree of the ability and desired binding ability of active constituent.It needs to apply The definite measurement of active constituent is determined in the judgement of practitioner, and different with each individual.It is suitble to first application and reinforces The scheme of injection is also variable, but is typically：First application, is followed by with 1 hour or a few houres interval by subsequently noting Penetrate or other application repeated doses.Alternatively, considering the continuous intravenous infusion for being enough to maintain the concentration in blood.

One embodiment considers that composition described herein is used to prepare the purposes of drug, and the drug is for treating herein Situation, disease or the illness.Can the physical trait based on patient/subject in need for the treatment of come compounding pharmaceutical, and can It is configured to single or multiple preparations with the stage based on situation, disease or illness.Can by drug packages in suitable packaging, The packaging has the label for being suitble to distribute to hospital and clinic, wherein the label, which is used to indicate treatment, has disease described herein The subject of disease.It can be by drug packages at single or multiple units.In the packaging that can be described below, including about combination The dosage of object and the specification of application.

It is also provided herein and is suitable for intravenous or intravitreal administration pre-filled note comprising preparation as described herein Emitter.Such precharging injection syringe can be packaged and mark for treating the relevant situation of angiogenesis, such as any Situation described in text.Packaging can further comprise the instruction for storing and applying.There is provided herein include preceding claims Any one of preparation the packaging for being suitable for intravenous or intravitreal administration one or more precharging injection syringes.Herein The preparation for additionally providing the drug containing preparation described herein is used to treat the relevant situation of angiogenesis, such as any Situation described in text.

Therapy

There is provided herein a kind of methods for treating subject (people is inhuman), pass through the preparation to subject's administration of antibodies, institute It states antibody and preferentially combines CD105.There is provided herein prevent or treat to give birth to angiogenesis/new vessels formation, excessive blood vessel At, tumour growth, tumor cell proliferation or thin vessels expand the method for related one or more diseases or illness, the method Including：Using the composition containing anti-CD105 antibody or its antigen-binding fragment, disease is thus prevented, treated, mitigates or reduced Or its severity.

When subject undergo disease S or S stagnation or partially or completely mitigate or reduce (specifically, including But it is not limited to the extension of survival period) when, realize the effective response of the present invention.It can monthly deposit to measuring expected get nowhere over year Live time, this depends on Prognostic Factors, including the stage of the number of recurrence, disease and other factors.Extend survival period include but It is not limited at least one moon (mo), about at least two moon, about at least three moon, about at least four moon, about at least six moon, about at least 1 year (yr), about at least 2 years, about at least 3 years, about at least 4 years, about at least 5 years etc. or in which any interlude.It can also Monthly to measuring total survival or progresson free survival over year.Alternatively, effective response can be, the sign or symptom or cancer of subject Disease load remains stationary and no longer deteriorate.Other instructions of the treatment of indication have been described in greater detail below.

The composition of antibody as described herein may be used as nontherapeutic agent (for example, as affinity purification reagent).In general, In one such embodiment, using conventional method known in the art, target protein is immobilized in solid phase (such as Sephadex resins or filter paper) on.The albumen of immobilization is set to contact the sample containing target (or its segment) to be purified Product, then wash support with suitable solvent, and the solvent can remove essentially all other than target protein in sample Material, the target protein is incorporated on the antibody of immobilization.Finally, with another suitable solvent, (such as glycine is slow Fliud flushing, pH 5.0) support is washed, the solvent will release target protein.Other than purifying, composition can be used for Detection, diagnosing and treating and the related diseases of CD105 and illness.

" contact " is defined herein as, make preparation provided herein physically close to cell as described herein, organ, The mode of tissue or fluid.Contact include any preparation provided herein applied systemically or topically, including but not limited in vitro, Internal and/or ex vivo internal program and method." combination " and " contact " uses interchangeably herein, and is intended to phase Same mode is defined.It can for example, by composition is administered to patient via any suitable way for vivo purposes To be in contact；The composition containing pharmaceutically acceptable excipient and carrier has been described in further detail above.

" prevention " used herein indicates to prevent, prevents the breaking-out of S or S, prevent it is related to angiogenesis or with The progress of the relevant disease of CD105 activity or illness.In one non-limiting embodiment, it is diagnosed as the patient of 1 phase cancer Preparation as described herein can be applied, thus prevents cancer progression to 2 phases.In another embodiment again, it is asymptomatic but Test, which is the patient of one or more biomarker for cancer positive, can apply preparation as described herein, thus prevent cancer Progress." inhibit (inhibition) " used herein, " treatment (treatment) " and " treating (treating) " interchangeably Use, and indicate, for example, the stagnation of S or S, the extension of survival period, the partially or completely improvement of S or S and The partially or completely elimination of tumour or transfer.

" subject " or " patient " is (for example, mammal such as people or non-human animal, such as primate, grinding tooth are dynamic Object, ox, horse, pig, sheep, camel, vigone etc.) can be the one or more clinics for showing disease or illness as described herein The mammal of performance and/or S or S.In some cases, subject can be asymptomatic, and still have institute State the clinical manifestation of disease or illness.It can be by the fusion protein on antibody conjugate to treatment part, or containing treatment part. It can be by the fusion protein on antibody conjugate to detectable part, or containing detectable part.In one embodiment, may be used With will be on antibody conjugate to both treatment part and detectable part.It can be by antibody conjugate to affinity tag (for example, purifying is marked Label) on, or recombinate ground with affinity tag (for example, purification tag) and be engineered.Affinity tag is conventional in the art.

Antibody provided herein or its segment so that they can be conjugated or be connected to treatment part and/or imaging moiety Or in detectable part and/or affinity tag.It is well-known in the art for conjugated or connecting peptides method.Compound Connection (in conjunction with) between label includes any mode known in the art, including but not limited to：It is covalently and non-covalent mutual Effect, chemically conjugated and recombinant technique.

" angiogenesis " all aspects herein for including blood vessel maintenance and development.Thus, angiogenesis includes： Lead to new capillary vascularization that new vessels are formed (no matter being formed or from pre-existing vascularization from new), Yi Jixian There are the maintenance and control of vascular system and thin vessels.Angiogenesis is a complicated process, which includes a series of successive The step of, including the vascular basement membrane of endothelial cell mediation and the degradation of interstitial matrix, the migration of endothelial cell, the increasing of endothelial cell Grow the formation with the capillary loops of endothelial cell.Angiogenesis includes growth and/or development (the also referred to as new green blood of new blood vessel Pipe is formed), the expanding of thin vessels, excessive or extended angiogenic growth and existing vascular system maintenance.

Term " the relevant disease of angiogenesis " is used herein to mean that certain pathologic processes of the mankind, medium vessels life At extending singularly.This further comprises：Angiogenesis situation and disease are such as made with associated angiogenesis, by angiogenesis At or with those of angiogenesis disease and situation.The non-limiting examples of such disease include：Various forms of cancers With transfer, macular degeneration and CNV.By combining CD105 and inhibiting angiogenesis, antibody as described herein can be used for treating blood Pipe generates relevant disease.

Term " anti-angiogenic therapy " is used herein to mean that targeted expression CD105 is (with static vascular system phase Than with higher horizontal expression in the vascular system in proliferation) cell and/or vascular system therapy；This is further wrapped It includes：For the therapy of angiogenesis (that is, leading to the new capillary vascularization that new vessels are formed), for existing vascular system And/or the therapy of excessive angiogenesis or angiogenic growth, for the therapy of thin vessels expansion, and for disease or the therapy of situation (for example, blood-vessels target therapy).The illustrative disease or situation considered within the present invention include but is not limited to：Various forms Cancer and transfer.

The relevant disease of angiogenesis as described herein can be, for example, cancer or transfer.In one embodiment, The cancer is solid tumor.Cancer to be treated includes, for example, the tumour based on epithelium.The cancer of for use such preparation for treating The non-limiting examples of disease include but not limited to lung cancer, gynecologic malignant tumor, melanoma, breast cancer, cancer of pancreas, oophoroma, Uterine cancer, colorectal cancer, prostate cancer, kidney, head cancer, cancer of pancreas, liver cancer (hepatocellular carcinoma), uterine cancer, neck cancer Disease, kidney (clear-cell carcinoma), sarcoma, myeloma and lymthoma.It can intravenously be applied for treating cancer or the preparation of transfer In patient.

In one aspect, using preparation, until the one or more marks of the relevant disease of angiogenesis or symptom mitigate.

For ophthalmic conditions, one or more S or Ss may include, but are not limited to vasoconstriction, inhibit with The relevant endothelial cell proliferation of eye disease, the muddy eyesight for the treatment of, provides stopping for visual loss at the S or S for removing bleeding Stagnant, improvement eyesight and/or prevention vascular leakage.

Term " recurrence ", " recurrence " or " recurrence " indicates, after clinical assessment disappearance of disease, cancer or disease are returned It returns.The diagnosis of far-end transfer or local recurrence can be regarded as recurring.

Term " maintenance therapy " indicates, the planned re-treatment given to help to maintain pervious therapeutic effect. Often give maintenance therapy assist in keeping cancer be in mitigate in or extend to the response of specific therapy (no matter progression of disease).

Term " progresson free survival " indicates in oncology, during therapeutic process and after, cancer do not grow when Between length.Progresson free survival includes：Patient have been subjected to the time of complete response or part response amount and patient Undergo the amount of the time of stable disease.

In one aspect, there is provided herein it is a kind of prevention or treatment subject cancer or transfer method, pass through by Any composition provided herein is administered to the patient with cancer or transfer.Such patient can be Symptomatic or without disease Shape.

In some cases, the service life of the treated patient of application extension of composition, reduction gross tumor volume, elimination are swollen Tumor, the Apoptosis or combination thereof for reducing cell Proliferation, increasing tumour cell.

If necessary, the method can also comprise：Surgical resection cancer and/or the additional anticancer of application Agent or treatment.Anticancer agent is provided in the other places this paper.

In one aspect, the S or S of the patient with cancer is improved.Improvement can be shown as, for example, pain The mitigation of pain, the reduction of tumor size, the elimination of tumour, the resistance that tumor size increases or the prevention of progression of disease, transfer are formed Only or metastatic growth inhibition or combination thereof.

In one aspect, the application of preparation as described herein reduce or eliminate patient to carry out surgical operation or with one kind or The demand of the treatment of a variety of additional antineoplastic agents or treatment.

Composition containing anti-CD105 antibody can be with the group containing anti-VEGF antibodies (or its antigen-binding fragment) Object is closed sequentially or simultaneously to apply.Such application includes but is not limited to following applications：In mutual about 12 weeks, at that In this about 8 weeks, in mutual about 4 weeks, in mutual about 3 weeks, it is interior at mutual about 2 weeks, interior at mutual about 1 week, In mutual 1 day, in mutual about 12 hours, in mutual about 6 hours, in mutual about 3 hours, mutual In about 1 hour, in mutual about 30 minutes, on the same day, at the same time or combination thereof.When the consideration present composition And/or combination treatment part multidose when, it should be understood that using commercially available product standard items, use base In the known dose and concentration of the age of subject, height, weight, health and other physical traits, can be empirically determined each From dosage.

Preparation can be administered to patient with therapeutically effective amount, the therapeutically effective amount passes through to be suitble to any therapeutic treatment Rational income/Hazard ratio inhibit disease or illness, effectively generate some desired therapeutic effects.In order to by the present invention's Preparation is administered to people patient, can carry out formulated by methods known to those skilled in the art.Therapeutically effective amount is in device The amount of desired therapeutic effect or preventive effect is realized at least partly in official or tissue.Realize disease or illness prevention and/ Or the amount itself of the anti-CD105 antibody or anti-VEGF antibodies needed for therapeutic treatment is unfixed.The amount of the antibody of application It can change with the type of disease, the extensive degree of disease and the size of mammal with disease or illness.At one In embodiment, with combinations of the above, two kinds of antibody described herein is administered to patient.Combined administration can refer in unitary system It is applied in agent or in separated preparation.

" application " herein indicate, by cause preparation in patient's body in a manner of, one or more preparations are supplied to Patient.Such application can be by any approach, including but not limited to：Locally, region or capapie, by subcutaneous , intravitreous, intradermal, intravenous, endarterial, in peritonaeum or intramuscular application (for example, injection).

The actual dose that active constituent in preparation can be changed is horizontal, and particular patient, preparation are effectively realized to obtain With the desired therapeutic response of mode of administration and the amount of the active constituent nontoxic to patient.The dosage level of selection will depend on a variety of Factor, including the discharge rate of the activity of the particular compound of use, administration method, administration time, the particular compound of use, Duration for the treatment of, other drugs, the compound being used in combination with the particular formulations used and/or material, patient to be treated Age, gender, weight, situation, general health and previous medical history and the well-known similar factor of medical domain.

Furthermore it is possible to one or more dosage of following administration of antibodies：2 times a week, 1 times a week, 1 time every 2 weeks, every 3 weeks 1 Secondary, every 4 weeks 1 time, it is 6 weeks 1 time every, 8 weeks 1 time every, 12 weeks 1 time every, 24 weeks 1 time or any Zhou Zuhe between them every.Also it examines Consider administration cycle, such as, for example, 1 times a week or 2 administration of antibodies, continue 2,3,4,5 or 6 weeks, then 1,2,3,4,5 Or it does not treat for 6 weeks.Alternatively, the response according to subject to treatment, the circulation time between treatment can be 1,2,3,4,5,6, 7,8,9,10,11 or 12 months.It is also contemplated for additional administration cycle within the present invention, including for example：Dosage described herein and The various combination recycled weekly.

Doctor or animal doctor can readily determine that and output the effective quantity (ED50) of the preparation of needs.For example, doctor or beast Doctor can since less than the dosage for realizing the required compound that is horizontal, using in the formulation of desired therapeutic effect, And dosage is gradually increased, until realizing desired effect.Alternatively, dosage can be kept constant.

By any convenient approach, preparation can be administered to patient by such as above-mentioned approach.The application no matter selected Approach, or by other conventional methods well known by persons skilled in the art, by the compound of the present invention, (it can be with suitable water The use of conjunction form) and/or preparation be configured to acceptable dosage form, all dosage forms described as follows.

The data obtained from cell culture test and/or zooscopy can be used for being formulated for the dosage range of the mankind. The dosage can change with the dosage form of use and the administration method used in the range.It, can be with for any compound Tentatively estimate treatment effective dose from cell culture test.Dosage can be prepared in animal model, be included in cell to realize The IC measured in culture₅₀The circulating plasma concentration range of (that is, realizing the concentration of the experimental compound of half maximum suppression).It can To measure the level in blood plasma, for example, passing through high performance liquid chromatography.Such information can be used for more accurately measuring Useful dosage in the mankind.Using any one of these methods, the preparation containing compound combination can also be assessed.

In one embodiment, the present invention considers to inhibit the angiogenesis in tissue.By a variety of methods, such as herein The method can evaluate the degree of the angiogenesis in tissue, and thereby evaluate the inhibition level realized.

The unique specificity of the antibody of identification (for example, preferential combine) CD105 or VEGF and inhibition angiogenesis, can be spy Sign is angiogenesis (new vessels are formed), thin vessels expansion, excessive angiogenesis, tumor cell proliferation and/or tumour life Long disease provides diagnosing and treating purposes.Antibody can be administered to various forms of cancers (primary tumor and turn Move) subject.

It should be appreciated that in addition to application preparation described herein other than, herein consider, can also use it is one or more additionally Angiogenesis inhibitors treat subject.

In order to illustrate the purpose of book and claims, term " angiogenesis inhibitors " has been used herein to mean that suppression The compound or molecule of the function of angiogenesis processed, including but not limited to：Peptide, albumen, enzyme, polysaccharide, oligonucleotides, DNA, RNA, recombinant vector and drug.Angiogenesis inhibitors are known in the art, consider all types herein.Compound and The non-limiting examples of molecule include：Natural and synthesis biomolecule, such as taxol, O- (chloracetyl-carbamyl Base) fumagine alcohol (" TNP-470 " or " AGM 1470 "), thrombin antithrombin III complex, thrombospondin -2, angiogenesis suppression The factor processed, the derivative angiogenesis inhibitors of human chondrocytes-(" hCHIAMP "), the derivative angiogenesis inhibitors of cartilage-, Platelet factor-4, gro- β, human interferon inducible protein 10 (" IP10 "), interleukin 12, Ro 318220, xanthic acid tricyclic Decyl- 9- base esters (" D609 "), Irsogladine, 8,9- dihydroxy -7- methyl-benzo [b] quinolizine bromide (" GPA 1734 "), Medroxyprogesterone, alpha-glucosidase inhibitors, genistein, Thalidomide, diamino-anthraquinone, removes at the combination of heparin and cortisone Green bristlegrass mycin, ursolic acid and oleanolic acid.The non-limiting examples of antibody include being directed to such as VEGF, vegf receptor or CD105 Different epitopes it is equimolecular those.In addition, the micromolecular inhibitor of vegf receptor is known, and consider herein.VEGF The non-limiting examples of acceptor inhibitor include ranibizumab, VEGF Trap, Sutent, Sorafenib, pazopanib, training plus Ni Bu and pazopanib.

The multiple combinations of these vegf receptor inhibitor can be applied together with preparation described herein.In an embodiment In, combination can cause lower dosage being used for the antibody or antigen binding.Such administration, which changes to be originated from, to be resisted The synergistic effect of body combination.

Cancer

CD105 is related with Tumor Angiongesis, and compared with the endothelium in normal structure, the quilt in the endothelium of various tumor tissues Consumingly raise.CD105 is raised in the tumor endothelial of wide scope.In addition, compared with corresponding normal structure, within the tumor There are stronger CD105 expression in skin.Thus, inhibit angiogenesis, the one kind for representing cancerous tumour to control with anti-CD105 antibody Treat selection.Preparation described herein can be used for treating cancerous tumour and transfer.The preparation can be used in the preparation of drug, The drug is for treating cancerous tumour and transfer.

VEGF represents a kind of target of antineoplaston, because its expression is raised in many solid tumors.VEGF is A kind of essential mediator of angiogenesis (going out new blood vessel from pre-existing angiogenic growth).The process is the growth of solid tumor Basis depends on the formation of new blood vessel.Certain small molecule therapy agent being capable of target vascular therapy endothelial growth factor receptor ("VEGFR")；This targeting of small molecule therapy agent can lead to anticarcinogenic effect.The reagent of vegf receptor is targeted by inhibiting new Vascularization blocks tumour growth indirectly.Inhibit the angiogenesis of VEGF inductions that can play anti-tumor effect or raising Anti-tumor effect, the VEGF without significantly inhibiting macrophage, osteoclast or chondroclast are stimulated.

Term " tumour " is used herein to mean that the cancerous tissue of expression CD105 and/or VEGF (just with same type The expression often organized is compared).Tumour may include solid tumor and half solid tumor.The non-limiting examples of tumour include：The white blood of people Disease, including non-T cell type (non-T) acute lymphatic leukemia (ALL), granulocytic leukemia；With human solid tumor and Half solid tumor, its peripheral vascular system express medium to high-caliber CD105 (the expression phases with the normal structure of same type Than), including angiosarcoma, breast cancer, gastric cancer, colon cancer, hodgkin lymphoma, lymthoma, glioblastoma multiforme (GBM), lung cancer, melanoma, myeloma, lymthoma, osteosarcoma, oophoroma, parotid tumor, pharynx cancer, prostate cancer, liver cell Cancer, kidney and proctosigmoid cancer.

Cancerous tissue to be treated is, for example, the endothelial tissue of the CD105 and/or VEGF of abnormal expression level.

In the case of the formation of the new vessels of not tumor tissues, tumor tissues will not obtain the nutrients of needs, raw Length slows down, and stops additional growth, degenerates, and finally becomes necrosis, leads oncogenic kill.There is provided herein by inhibiting swollen Tumor angiogenesis inhibits the method for tumor angiogenesis.Similarly, there is provided herein the methods for inhibiting tumour growth.

The formation of the method confrontation transfer is also particularly effective, because their formation needs the blood of primary tumor Pipe generates so that metastatic carcinoma cell may exit off primary tumor, and they need new vessels in the foundation of second position It is formed to support the growth of transfer.

It should be understood that " subject for suffering from cancer/transfer " of the present invention can express mutain, (tumour correlation is anti- It is former) or mutator, and the still not no symptom of disease.In a non-limiting examples (itself and the saltant type K-ras of colon cancer Albumen is related) in, the subject with saltant type K-ras albumen is subject to be treated in some colon cells, even if The subject may the still not no symptom of colon cancer." S or S of disease " represents the performance of disease clinically identified Or indication.

The subject of " treatment " with tumour or transfer indicates that after treatment, the S or S part of subject subtracts Gently, complete mitigation or remains stationary.The patient treated can show the partially or completely mitigation of tumor load.This is intended to Including preventing, treating and curing.In one non-limiting example, treatment suffers from height metastatic cancer (for example, breast cancer) Subject, wherein additional transfer does not occur, or compared with no subject for receiving treatment, the number of transfer is reduced. In another non-limiting examples, treat subject, wherein the solid carcinoma of subject size reduce, or with do not receive The subject for the treatment of compares, and the size of solid carcinoma does not increase.In another non-limiting examples, with no receiving treatment The number of the cancer cell of subject is compared, and the number of the cancer cell in the subject for the treatment of does not increase or number is reduced.Also may be used It is defined as with that will improve, for example, the reduction of cell Proliferation, the reduction of cell number, the increase of Apoptosis and/or treated The increase of the survival period of subject.

It stays in the tumour treated in method described herein or cancer includes but is not limited to：Lung cancer, gynecologic malignant tumor, Melanoma, breast cancer, the cancer of the brain (for example, glioblastoma multiforme, " GBM " or glioma), cancer of pancreas, ovary Cancer, uterine cancer, colorectal cancer, prostate cancer, kidney, head cancer, liver cancer (hepatocellular carcinoma), neck cancer, kidney (clear-cell carcinoma), the moon Stem cancer, gastric cancer, thyroid cancer, carcinoma of urinary bladder, sarcoma, cancer, myeloma and lymthoma.In one embodiment, to be treated swollen Tumor is solid tumor or half solid tumor.In another embodiment, tumour to be treated is primary tumor.In another implementation In scheme, tumour to be treated is metastatic tumo(u)r.In one embodiment, tumour or cancer to be treated belong to epithelium and rise Source.In another embodiment, cancer to be treated is myeloma.In another embodiment, cancer to be treated is Oophoroma.In another embodiment, cancer to be treated is kidney/renal cancer.In another embodiment, to be treated Cancer is hepatocellular carcinoma/liver cancer.

As needed, compound can be jointly applied with one or more additional therapeutic treatments, including but unlimited In：Doxorubicin, cyclophosphamide, taxol, pemetrexed, Temozolomide, oxaliplatin, Cetuximab, Victibix, Suo La Non- Buddhist nun, Sutent, Gefitinib, Tarceva, 5 FU 5 fluorouracil, Irinotecan, Hycamtin, folinic acid, Bortezumib, lenalidomide, Thalidomide, capecitabine, docetaxel and many other common cancers as described herein are treated Method." radiation " used herein indicates, for example, microwave, ultraviolet light (UV), infrared ray (IR) or α-, β-or γ-radiation.It uses Routine techniques, " can concentrate " or locally delivering radiates, and will radiate the one or more tumor locus of targeting, and non-radiating is whole Body body.It should be appreciated that the list for the therapeutic scheme being listed below represents routine treatment, but the present invention is included herein not There is specifically disclosed other known therapeutic scheme.

In one embodiment, the cancer is oophoroma, and one or more additional therapeutic treatments are outer Section's operation, chemotherapy are (for example, Doxorubicin, Doxil, gemcitabine and the chemotherapeutant based on platinum Such as cis-platinum, carboplatin and oxaliplatin), melphalan, topoisomerase I inhibitor such as Hycamtin and Irinotecan, be based on The therapy of taxane, hormone, radiation-therapy, total body hypothermia, isoflavone derivative, the macrolides of cytotoxicity are such as angstrom rich Mycin, angiogenesis inhibitors such as bevacizumab, signal transduction inhibitor such as trastuzumab, gene therapy, RNAi therapies, Immunotherapy, monoclonal antibody, phosphatidylinositols sample kinase inhibitor such as rapamycin or any combination of them.This paper institutes The combination therapy of the antibody stated and oophoroma therapy can also provide the lower dosage of any therapy or the two, this is because altogether With the synergistic effect of the application therapy.

In one embodiment, the cancer is kidney/renal cancer, and one or more additional therapeutic treatments It is surgical operation, chemotherapy, pazopanib, interferon-' alpha ' or IL-2.In another embodiment, the additional agents are Vegf receptor inhibitor.The non-limiting examples of vegf receptor inhibitor include the above, VEGF Trap, Sutent, Sorafenib, pazopanib and pazopanib.The combination therapy of antibody as described herein and kidney therapy can also provide any The lower dosage of kind therapy or the two, this is because the synergistic effect of the therapy is co-administered.

In one embodiment, the cancer is myeloma, and one or more additional therapeutic treatments are Surgical operation, radiotherapy, bortezomib, lenalidomide or Thalidomide.The dosage of any one of these therapies is ability Known to domain, and can correspondingly it be adjusted in combination therapy.

In one embodiment, the cancer is prostate cancer, and one or more additional therapeutic treatments It is surgical operation, radiotherapy (for example, external beam or brachytherapy), (androgen inhibits hormonal deprivation, including uses Abiraterone), heat shock protein 90 (HSP90) inhibitor, chemotherapy (for example, docetaxel, Estramustine, based on platinum Chemotherapy such as cis-platinum, carboplatin, Satraplatin and oxaliplatin), prednisone or prednisolone, reduce the drug of cholesterol such as he Spit of fland class drug, luteinizing hormone-releasing hormone (LHRH) agonist, RNAi therapies, the therapy based on dendritic cells, genetic modification At secretion granulocyte-macrophage-colony-stimulating factor (GM-CSF) full tumour cell (also referred to as GVAX) or theirs is any Combination.In another embodiment, the additional agents are vegf receptor inhibitor.Vegf receptor inhibitor it is non-limiting Example includes：VEGF Trap, Sutent, Sorafenib, pazopanib and pazopanib.

In one embodiment, the cancer is lung cancer, and one or more additional therapeutic treatments are outer Section operation, radiotherapy (for example, chest radiotherapy, using the radiation-therapy of charged particle, uracil-Tegafur and be based on platinum Chemotherapy (for example, cis-platinum, carboplatin, oxaliplatin etc.) and vinorelbine, Tarceva, Gefitinib, anti-epidermal growth Factor receptor antibody (for example, Cetuximab), the micromolecular inhibitor of tyrosine kinase, participation proliferation of lung cancer cells are related to Direct inhibitor, aurora kinase inhibitors, the heat therapy of induced with laser, RNAi therapies, the genetic modification of albumen are thin at secretory granule The full tumour cell (also referred to as GVAX) or any combination of them of born of the same parents' Macrophage-Colony stimulating factor (GM-CSF).Additionally Therapeutic treatment include taxol or pemetrexed.In another embodiment, the additional agents are vegf receptor suppressions Preparation.The non-limiting examples of vegf receptor inhibitor include VEGF Trap, Sutent, Sorafenib, pazopanib and pa Azoles pa Buddhist nun.The dosage of any one of these therapies is known in the art, and can correspondingly be adjusted in combination therapy Section.

In one embodiment, the cancer is breast cancer, and one or more additional therapeutic treatments are Surgical operation, monoclonal antibody (for example, Her-2 antibody, Trastuzumab), adjuvant chemotherapy such as single agents chemotherapy or Combination chemotherapy (for example, Polychemotherapy based on anthracycline antibiotic and based on taxane) or target-specificity Trastuzumab (be with or without endocrine operation, be with or without PMRT), vinorelbine), doxorubicin, cyclophosphamide, Ka Peita The estrogen receptor tune of shore, docetaxel, selective estrogen receptor modulators such as tamoxifen and Raloxifene, allosteric Agent such as Trilostane, radiation are saved (for example, interstitial brachytherapy, balloon-system, three-dimensional conformal external radiation and surgery hand Radiotherapy in art), inhibit whole body synthesis aromatase inhibitor (for example, Anastrozole, Exemestane and Letrozole), RNAi therapies, inhibitive ability of immunity and antiproliferative rapamycin intravenous analog such as tamiros or theirs is any Combination.The summary of the method for tissue-culture model outside said three-dimensional body for carrying out breast cancer, description exist：Kim et al., Breast Cancer Research Treatment 85(3): 281-91(2004).Other internal and external moulds for testing cancer Type is known, and can be used for testing antibody as described herein.In another embodiment, the additional agents are VEGF Acceptor inhibitor.The non-limiting examples of vegf receptor inhibitor include：VEGF Trap, Sutent, Sorafenib, Ah former times replace Buddhist nun and pazopanib.The dosage of any one of these therapies is known in the art, and can in combination therapy correspondingly It is adjusted.

In one embodiment, the cancer is colon cancer, and one or more additional therapeutic treatments are Surgical operation, radiation-therapy and chemotherapy are (for example, 5 FU 5 fluorouracil (5-FU), levamisol, folinic acid or Semustine (Methyl CCNU)), N- [2- (dimethylamino) ethyl] acridine-4-carboxamide carboxylic acid amides anticarcinogen related with other；Non- topology Isomerase II inhibitor, Irinotecan, Liposomal topotecan, taxanes anticancer agent (for example, taxol or docetaxel), Xanthone acetate type compound is (for example, 5,6-Dimethylxanthenone-4-acetic acid (5,6-dimethylanthenone-4- Acetic acid) PMAA), laminarin, site-selective ring AMP analogs (for example, 8- chlorine adenosine 3', 5'- ring phosphorus Hydrochlorate), the Pyranoindole inhibitor of Cox-2, the carbazole inhibitor of Cox-2, the tetrahydro carbazole inhibitor of Cox-2, Cox-2 Indene inhibitors, NSAIDS local inhibitor (for example, ortho-aminobenzoic acid, aspirin (5- acetylsalicylic acid), Olsalazine Sodium, carbon heterocyclic acid, Carprofen, Chlorambucil, double chloroaniline phenol acetic acid, fenbufen, fenclofenac, fenoprofen, Flufenamic acid, Flurbiprofen, Fluprofen, frusemide, sodium aurothiomalate, brufen, Indomethacin, indoprofen, Ketoprofen, lonazolac, Lip river Suo Luofen, Meclofenamic Acid, mefenamic acid, melphalan, naproxen, penicillamine, phenylacetic acid, propionic acid, salicylic acid, willow nitrogen sulphur pyrrole Pyridine, tolmetin, buprofezin phenylbutazone propazine, NSAID, Meloxicam, former times health class, piroxicam, takes pyridine, pyrrole sieve at sulindac Former times health β ring glucan, tenoxicam, Etodolac and olsapozine), the inhibitor of HER-2/neu, RNAi therapies, GM-CSF, Monoclonal antibody (for example, anti-Her-2/neu antibody, anti-CEA antibody, A33 (HB 8779), 100-210 (HB 11764) and 100-310 (HB 11028)), Cetuximab, Victibix, hormonotherapy, aminopyrimidine, camptothecin derivative (for example, CPT- 11), folinic acid (FA), gemcitabine, Ara-C, the chemotherapy based on platinum such as cis-platinum, carboplatin and oxaliplatin, The phosphodiesterase inhibitors or any combination of them of cGMP- specificity.In one embodiment, the additional treatment Property processing be 5 FU 5 fluorouracil, folinic acid and oxaliplatin combination (FOLFOX).In one embodiment, described additional Therapeutic treatment is the combination (IFL) of 5 FU 5 fluorouracil, Irinotecan and folinic acid.In one embodiment, described additional Reagent is Cetuximab.In one embodiment, the additional agents are Victibixes.In another embodiment, The additional agents are vegf receptor inhibitor.The non-limiting examples of vegf receptor inhibitor include：VEGF Trap, Buddhist nun of relaxing replace Buddhist nun, Sorafenib, pazopanib and pazopanib.The dosage of any one of these therapies is known in the art, and can be with It is correspondingly adjusted in combination therapy.

In one embodiment, the cancer is cancer of pancreas, and one or more additional therapeutic treatments are Surgical operation, radiation-therapy, 5 FU 5 fluorouracil and radiotherapy, constitutional treatment, holder, gemcitabine, gemcitabine and radiation Therapy, Cetuximab, Tarceva, chemicotherapy or any combination of them therapeutic treatment combination.In another reality It applies in scheme, the additional agents are vegf receptor inhibitor.The non-limiting examples of vegf receptor inhibitor include： Afliberceipt, Sutent, Sorafenib, pazopanib and pazopanib.

It can be at one or more different time points, before being included in therapeutic scheme, in the process and later, about sign Or symptom assesses patient.Treatment can lead to the situation for improving subject, and can be by determining whether to have occurred and that One or more in following factors are assessed：The reduction of tumor size, the reduction of cell Proliferation, cell number subtract Less, the increase for the reduction, Apoptosis that new vessels are formed or at least part form the survival of the cell of cell proliferative diseases Phase reduces.In some cases, one or more of these events can lead to partially or completely elimination and/or the patient of cancer The extension of survival period.Alternatively, for Terminal cancer, treatment can lead to stagnation, better quality of life and/or the survival period of disease Extension.

When sequential application composition, including the composition of anti-CD105 antibody as described herein can be with for example, anti- It is applied before or after VEGF antibody (or its antigen-binding fragment).

When composition is administered simultaneously, the composition containing anti-CD105 antibody can with contain anti-VEGF antibodies The identical position of composition or the application of different positions.

In another embodiment, there is provided herein contain anti-CD105 antibody and anti-VEGF antibody (or its antigen knot again Close segment) composition (drug), the one or more bioactivity of CD105 and VEGF respectively can be inhibited, such as promote to have Mitotic activity, cell Proliferation, tumour growth, new vascular generation or angiogenic activity.

It should be appreciated that therapeutic scheme may include each applied in one or many compositions described herein.It can be with Composition is applied in single dose or multidose.The application of separated composition can by identical approach or be passed through Different approach.

In one embodiment, every 1 to the 3 week application of composition, continues 6 to 12 periods, or until tumour progression.Institute The method of stating may further include for every 1 to 12 all the step of lasting up to 2 years using each composition.It is non-limiting at another It applies and is happened at the 1st week in example, while anti-CD105 antibody and anti-VEGF antibody (or its antigen-binding fragment), then exist 1st, 2,3 or 4 week additional applying said compositions, repeat 6 to 12 periods, or until tumour progression wherein being administered simultaneously, and Then every 1 to 12 all applying said compositions last up to 2 years.

In a non-limiting examples of the method for treating the cancer in patient, the method includes：Operation is cut Continue 12 months except cancer, and in the 1st to the 3 anti-CD105 antibody of all applications and anti-VEGF antibody (or its antigen-binding fragment), or Until tumour progression, then in the anti-CD105 antibody and anti-VEGF antibody that dosage is administered simultaneously the 1st to 12 week, (or it is anti- Former binding fragment).In addition, while anti-CD105 antibody and anti-VEGF antibody (or its antigen-binding fragment) apply can with every 1 to It repeats within 3 weeks to last up to 6 weeks.Optionally, anti-CD105 antibody were applied the method further includes each to 12 weeks and resisted VEGF antibody (or its antigen-binding fragment) lasts up to 2 years.It should be understood that therapeutic scheme can be with prison provided herein Survey method combines, to determine and if when to need anti-CD105 antibody and anti-VEGF antibody (or its antigen using extra dose Binding fragment).

Combination therapy can provide synergistic effect and/or beneficial effect, or can allow lower unitized dose, to provide The safety margin of bigger.The present invention includes such therapeutic scheme：It enhances anti-CD105 antibody and anti-VEGF (or its antigen Binding fragment) for preventing, controlling, treating or eliminating cancer or the preventive effect or therapeutic effect of other diseases.

In one embodiment, additional therapeutic treatment is applied to subject, such as, for example, Agiogenesis inhibition Agent (as described herein).Composition containing such additional therapeutic treatment can combine Internet of Things with as described herein other Close ground (sequentially or simultaneously) application.

Provided herein in a kind of non-limiting method for the treatment of cancer, additional therapeutic treatment includes：Outside Section's operation excision cancer, irradiation, one or more chemotherapeutics or combination thereof, and apply parallel one or more described herein Composition.In one aspect, the application of composition can be, for example, 20 minutes intravenous infusions.

It is related to the ophthalmology disease of angiogenesis

In one aspect, the present invention provides one or more preparations provided herein by applying therapeutically effective amount to patient It is green to treat diabetic retinopathy, macular degeneration, choroidal neovascularization formation, macular edema and/or neovascular The method of light eye.

Macular degeneration (AMD) is the light in a part for the central retina (being referred to as macula lutea) for being responsible for fine definition vision The forfeiture of receptor.Macular degeneration and extracellular matrix component and other fragments are between retinal pigment epithelium and blood-vessels film Film in abnormal deposition it is related.The fragment-sample material is referred to as drusen.By ophthalmoscope eye examination, drusen is observed. Normal eye may have the macula lutea of not drusen, and drusen may largely be present in retinal periphery.Not any In the case that macular vision loses, presence of the soft glass wart in macula lutea is considered the AMD of early stage.The feature of macular degeneration exists (CNV) is generated in choroidal neovascular, i.e., forms abnormal vascular below the retinal pigment epithelium of retina (RPE) layer. These blood vessels break through Bruch's membrane, destroying retinal pigment epithelium, bleeding, and ultimately cause macula lutea cicatrization, and the latter causes The central light loss of depth (disciform scar is formed).

Other than other ophthalmology diseases, choroidal neovascular generates (CNV) and is common in macular degeneration, and and choroid The proliferation of endothelial cell, the excess generation of extracellular matrix are related with the formation of fibrovascular Subretinal membranes.On retinal pigment The generation of epithelial cell proliferation and angiogenesis factor seems that influencing choroidal neovascular generates.

Diabetic retinopathy (DR) is that the ophthalmology disease for being characterized in that excessive angiogenesis is formed in diabetes, The reason is that capillary basement membrane thickens and lacks the contact between the pericyte of capillary and endothelial cell.Pericyte The leakage for increasing capillary is lost, and leads to the destruction of blood-retinal barrier.Diabetic retinopathy is that capilary regards The result of nethike embrane variation.The peripheral cells death of hyperglycemia induction and thickening for basement membrane, lead to the insufficiency of vascular wall.This It is a little to destroy the formation for changing blood-retinal barrier, and retinal vessel is also made to become permeability higher.Thin vessels (such as eye Those of in) it is particularly vulnerable to the influence that the blood glucose (blood-glucose) of difference controls.Glucose and/or fructose build up damage Tiny blood vessels in retina.When on impaired blood vessels leak fluid and lipid to macula lutea, macular edema can also be formed.This A little liquid make macula lutea swelling, the latter make eye-blurred.The destruction also leads to the anoxic at retina.

With progression of disease, the anoxic in retina is stimulated along retina and in clear gel sample vitreous humor Angiogenesis in (it is filled within the eye).If be not treated in time, these new blood vessels can make eye-blurred, and break with bleeding Bad retina.Fibrovascular proliferation can also cause traction detachment of retina.New blood vessel can also grow the angle into camera oculi anterior In, and cause neovascular glaucoma.

Proliferative vitreoretinopathy in hyaloid membrane and the cell membrane on the surface of retina and fibre The cell Proliferation for tieing up modified film is related.Retinal pigment epithelium cell proliferation and migration are common in the ophthalmology disease.With it is Hypertrophic The related film of vitreoretinopathy contains extracellular matrix component such as collagen I, II and IV types and fibronectin, and gradually becomes Obtain fibrosis.

In developed country, age-related macular degeneration (AMD) and diabetic retinopathy are that 2 of blindness are main Reason.VEGF Trap, ranibizumab and piperazine Jia Tani have been improved for the available treatment option of AMD patient.Ranibizumab It is Fab, and VEGF Trap is fusion protein.Both of which combination vascular endothelial growth factor (VEGF), and so far It is rendered go out most it is mirable treatment AMD result；But the patient experience visual acuitys of only a small number of treatments is obviously improved. The anti-angiogenic therapy in the target other than VEGF is focused on, can be overcome related with the reagent for targeting VEGF pathway Some limitations.

Anti- CD105 antibody as described herein can be used for treating or preventing macular degeneration, CNV, diabetic retinopathy Change or proliferative vitreoretinopathy.This document describes become via application Antybody therapy as described herein or prevention macula lutea Property, the method for CNV, diabetic retinopathy, macular edema or proliferative vitreoretinopathy.It is as described herein anti- CD105 antibody can also vasoconstriction, inhibition and the relevant endothelial cell proliferation of eye disease, remove bleeding sign or disease Shape, the stagnation for providing visual loss, improves eyesight and/or prevents vascular leakage the muddy eyesight for the treatment of.It is as described herein anti- CD105 antibody can be used for treatment macular degeneration, CNV, diabetic retinopathy, macular edema or hyperplastic vitreous and regard In the drug of retinopathy.

In addition, anti-CD105 antibody as described herein can also with treatment macular degeneration, CNV, diabetic retinopathy, The known therapies and/or compound combination of macular edema or proliferative vitreoretinopathy use.Such compound Example includes, but are not limited to ranibizumab, VEGF Trap and piperazine Jia Tani.It, can be with other than administration mode as described herein The anti-CD105 antibody is applied by intravitreous.The non-limiting examples of intravitreal administration pattern include glass The use of internal injection and intravitreal implant.

The improvement of patient and the response to treatment can be assessed.Treatment includes but is not limited to：Mitigate macular edema, the faces CNV The increase of long-pending reduction and visual acuity.The measurement of S or S is as it is known in the art, and in the examples below furtherly It is bright.

According to the embodiments described herein, preparation as described herein can be administered alone, or with it is one or more additional Activating agent or inert agents be administered in combination.When using combining, anti-CD105 antibody and anti-VEGF antibody (its antigen can be used Binding fragment) while or sequential application.

Function test

Preparation as described herein can be assessed in a variety of external, internal and ex vivo experiments.This field can be used It is any suitable known to technical staff to test to monitor such effect.This document describes several such technologies.

The measurement of CD105 signal transductions and function

CD105 (endothelial factor) is by a member of the TGF-β receptor family of the endothelial cell expression in being proliferated, endothelial cell Proliferation needs the CD105 of normal level.CD105 strong expressions in the angiogenesis vascular system of solid tumor are related to blood vessel life At/vascular development, and it is complementary transforming growth factor β (TGF-β) receptor.CD105 is one kind in leukaemia cell and Nei The homodimer glycoprotein expressed on chrotoplast.2 kinds of isotype L- endothelial factors (170 of CD105 are characterized KDa) and S- endothelial factors (160 kDa), they the difference is that the amino acid sequence of their cytoplasmic tail.

CD105 expression increases (passing through the generation of hypoxia inducible factor-1-α (HIF-1- α)) by hypoxic, and protects low Oxygen cell is from Apoptosis.The effect of CD105 is that the signal for a variety of kinases receptors compounds for adjusting TGF-β superfamily passes Lead, the TGF-β superfamily include TGF-β receptor (TGF-β R), Activin receptor-sample kinases (ALK) and activator protein by Body.In the presence of no CD105, the activation of TGF-β receptor causes the phosphorylation of SMAD albumen, the latter that endothelial cell is inhibited to give birth to It is long.But TGF-β adjusts SMAD protein phosphorylations to the activation of CD105.Final result is that TGF-β receptor activation is thin to endothelium The release of the growth inhibition effect of born of the same parents.

The prevention that anti-CD105 antibody activates CD105 synergistically plays a part of to inhibit endothelial cell growth with TGF-β. TGF-β can stimulate 2 kinds of unique I receptors/SMAD signal transduction paths with adverse effect in endothelial cell.TGF- β/ALK5 signal transduction paths (A) lead to the inhibition of cell Proliferation and migration, and TGF-β/ALK1 approach (B) inducing endothelial cell Proliferation and migration.The CD105 (a kind of complementary TGF-β receptor) highly expressed in angiogenesis is that ALK1 signals pass Necessary to leading.In the presence of no CD105, TGF-β/ALK5 signal transductions are dominant, and maintain static endothelium.It is high CD105 expression stimulation ALK1 approach, and inhibit ALK5 signal transductions indirectly, to promote the state of activation of angiogenesis.

It in one non-limiting embodiment, can be anti-to assess about inhibition angiogenesis and endothelial cell proliferation Body.The combination of anti-CD105 antibody and HUVEC will not prevent TGF-β and HUVEC later in conjunction with.Thus, anti-CD105 is anti- The direct inhibition of body Endothelial Cells Growth represents the root of the anti-angiogenesis effect and tumor inhibitory effect observed in vivo One of present mechanism.In another embodiment, can about block angiogenesis (by prevent Smad1/5/8 phosphorylations and/ Or signal transduction) assess antibody.CD105 participates in promoting angiogenesis, the letter by the signal transduction of TGF-β/ALK1 Number conduction further relate to Smad2/3 albumen phosphorylation reduction and/or blocking.It in another embodiment, can be about resistance Angiogenesis (passing through enhances Smad2/3 phosphorylations and/or signal transduction) break to assess antibody.

For measuring antibody provided herein to the phosphorylation of TGF-β/ALK1 signal transduction paths and/or Smad1/5 It blocks or the methods and techniques of inhibiting effect includes but is not limited to：Known molecular engineering.As example, using to TGF-β/ The Western blotting of any one of ALK5 or TGF-β/ALK1 approach antibody of protein-specific is determined for public herein Inhibiting effect and/or stimulation of the anti-CD105 antibody opened to TGF-β/ALK5 or TGF-β/ALK1 approach.Similarly, The detection of the mRNA of TGF-β/ALK5 or the albumen of TGF-β/ALK1 approach or the adjusting of mRNA are participated in, can be used for measuring herein The inhibiting effect and/or stimulation of disclosed antibody.Cell for measuring TGF-β/ALK5 or TGF-β/ALK1 approach is believed Number conduction other methods be known in the art, and herein consider.

Using experiment well known in the art, for example, by combining experiment such as ELISA, competitive ELISA, surface plasma Resonance body and influence (being described in greater detail below) to HUVEC cells, it can be estimated that anti-CD105 antibody disclosed herein Activity.

SCID/nude mice

A kind of method for measuring tumour growth uses SCID mice as follows：People's M21 melanoma cells of subconfluent are harvested, Washing, is resuspended in sterile PBS (20 x 10⁶/mL).100 μ L M21 human melanins are hypodermically injected to SCID mice Oncocyte (2 x 10⁶) suspension.3 days after tumor cell injection, mouse is not treated, or with one or more controls or examination It tests preparation intravenously or peritonaeum treats mouse (for example, 100 μ g/ mouse) interiorly.Treatment mouse daily, continues 24 days.It uses Caliper measures tumor size, and uses formula V=(L x W²Volume is estimated in)/2, and wherein V is equal to volume, and L is equal to length, W Equal to width.

A kind of method for measuring tumour growth uses nude mice as follows：By the MDA-MB-435 tumours in 50 μ l PBS Cell (0.4 × 10⁶Cell/mouse) it is implanted into normotopia in the mammary fat pad of Female nude mice (5-6 week old).When tumour reaches big About 50-80 mm³Average external volume when, by mouse be randomized (at least 10/group), and begin to use following dosage one kind or Treatment in the intravenous or peritonaeum of Multiple Antibodies, 2 times a week：1 μ g (0.05 mg/kg)/agent in 100 μ l PBS, 10 μ g (0.5 mg/kg), 100 μ g (5 mg/kg) or 100 μ of 200 μ g (10 mg/kg) or 100 μ g control antibodies or medium PBS l.In some researchs, non-treatment group can also be evaluated.Tumor size is measured using caliper, and uses formula V=(L x W²Volume is estimated in)/2, and wherein V is equal to volume, and L is equal to length, and W is equal to width.

BALB/c Syngeneic mouse models

Alternatively, can also using BALB/c Syngeneic mouse models come assess tumour growth and antibodies on tumor described herein growth Inhibit, such as such as Tsujie et al.,Int. J. Oncology, 29:1087-1094 (2006) institute illustration.

Mouse

Another test measurement gomphosis mouse:Angiogenesis in people's mouse model, and it is referred to as chimeric mouse assay.The examination It tests and is described in detail via other people, and be further described herein for measuring angiogenesis, new green blood Pipe is formed and the recession of tumor tissues.Referring to Yan, et al. (1993)J. Clin. Invest. 91:986-996。

Chimeric mouse assay is the useful test model that body vessel generates, because the skin graft of transplanting is being organized Normal application on human skin is very similar on, and the new vessels formation entirely organized is occurring, wherein actual people's blood Pipe is grown to from the application on human skin of transplanting in the human tumour tissue on the surface of the application on human skin of transplanting.By with human specific Endothelial cell marker to the immunohistochemical staining of neovasculature, it can be verified that new vessels are formed in people graft In source.

Chimeric mouse assay confirms the recession that new vessels are formed, this amount and recession degree based on new blood vessel growth.This Outside, the influence of the growth to being implanted in any tissue (such as tumor tissues) on the skin of transplanting can easily be monitored.Most Afterwards, the experiment is useful, because there are the internal contrasts of toxicity in the pilot system.Gomphosis mouse is exposed to and is appointed What test reagent, therefore the health of mouse can indicate toxicity.Other animal models as described herein and known in the art also may be used For in method described herein.

Rabbit eye assay

Another measurement of angiogenesis is internal lagophthalmos model, and is referred to as rabbit eye assay.The rabbit eye assay is via it Other people are described in detail, and have been used for measuring angiogenesis in the presence of angiogenesis inhibitors and new green blood Pipe is formed, such as D ' Amato et al. (1994)Proc. Natl. Acad. Sci. USA, 91(9):4082-4085 institutes illustration 's.

Rabbit eye assay is the test model that generally acknowledged body vessel generates, can be with because by the native Zona cornea of eye New vessels forming process is easily observed, the process is by rabbit blood vessel from growth of the edge of cornea into cornea come illustration.In addition, The degree and amount of the stimulation of new vessels formation or the recession of inhibition or new vessels formation can be easily monitored at any time.

Finally, rabbit is exposed to any test reagent, hereafter the health of rabbit can indicate the toxicity of experiment reagent.

In brief, it carries out chicken CAM (CAM) to test, contains one or more controls or experiment chemical combination in implantation 48 hours after 0.5% carboxymethyl cellulose granule of object, the influence to developmental vascular system is recorded.By the poly- (methyl being implanted into Hydroxy-ethyl acrylate) granule (Hydron;Interferon Sciences, New Brunswick, NJ) induce cornea new Angiogenic, the granule contain 650 ng and ulcerlmin (sucrose aluminium sulfate;Bukh Meditec, Copenhagen) it combines Effective angiogenic proteins basic fibroblast growth factor (bFGF).Addition of the ulcerlmin into granule, protection BFGF and provides its slow release from degradation, to generate consistent aggressive angiogenesis, than individual bFGF/ The angiogenesis of Hydron inductions is more notable.Up to 4 days after forming granule, bFGF can be detected from containing sulphur in vitro The release of the granule of sugared aluminium/Hydron, in contrast to this, for containing only the granule of Hydron, only 1 day.Prepare granule as follows：It is mixed Close brine and 40 mg ulcerlmins that 110 μ l contain 12 μ g recombinant bfgfs (Takeda, Osaka)；80 μ l are added in the suspension In 12% (wt/vol) Hydron in ethanol.Then the aliquot of the mixture (10 μ l) is pipetted into teflon Chinese toon On (Teflon pegs), and allow drying, generates about 17 granules.

By in the micro- bag of cornea of every eye of the female New Zealand white rabbits of granule implantation anesthesia, 2 mm of isolated edge then exists Single local application erythromycin ointment on the surface of cornea.Histological examination in continuous several days confirms in cornea to granule gradually Into property angiogenic growth, rare inflammatory cell is only observed.It will not change the blood vessel using the serious immunosupress of whole body irradiation Response is generated, the granule for containing only ulcerlmin will not induction of vascular generation.New blood vessel is mainly induced by bFGF (rather than inflammation). From implantation 2 days later, the one or more chemical combination being suspended in daily to detoxification by gastric lavage in 0.5% carboxymethyl cellulose Object or individual medium.Before it will be implanted into granule, the total body radiation of 6 Gy of immunosuppressive animal receiving 6 minutes.It should The radiation of dosage causes significant white blood cell to reduce, and is reduced to the 2nd day white blood cell count(WBC)>80%, subtract to the 3rd day white blood cell count(WBC) It is few>90%, the result is consistent with pervious report.

Identical cornea specialist (M.S.L.) is every other day with masking with slit lamp examination animal.By with point The clock hour number (C) that plate measures the length of vessel (L) and the edge being related to of isolated edge is drawn, determines corneal neovascularization Area.The area of endless belt section is determined using formula：C/12 x 3.1416 [r² - (r - L)²], the mm of wherein r=6, The radius of the rabbit corneal measured.The uniformly continuous band that the new vessels of neighbouring granule are formed is measured, therefore, it is possible to assess new life Total inhibition of vascularization.

Mouse stromal plug angiogenesis generates experiment

In order to confirm influence of the preparation to angiogenesis, mouse stromal plug angiogenesis can be used to generate experiment.By various growths The factor (for example, IGF-1, bFGF or VEGF) (250 ng) and heparin (0.0025 unit/mL) and the growth factor described in the pastReductionMatrigel mix (Montesano, et al.,J. Cell Biol. 1983, 97:1648-1652; Stefansson, et al.,J. Biol. Chem. 2000, 276:8135-8141).Can by preparation as described herein or Control antibodies are included in matrigel product, wherein using one or more dosage groups of animal.In control experiment, do not having In the presence of growth factor, matrigel is prepared.0.5 mL matrigel products are hypodermically injected to mouse, and allow to be incubated 1 week.It is incubating After educating the period, mouse is put to death, the matrix rubber plug of polymerization is taken out by surgical operation.It is (including immune by 2 kinds of determining methods Tissue chemical analysis and content of hemoglobin), it is quantitative in matrix rubber plug angiogenesis (Furstenberger, et al.,Lancet. 2002, 3:298-302;Volpert, et al.,Cancer Cell2002, 2(6):473-83；And Su, Et al.,Cancer Res. 2003, 63:3585-3592).For immunohistochemical analysis, matrix rubber plug is embedded in OCT In, it is rapidly frozen, and prepare 4 μm of slices.In methanol/acetone (1:1) slice of fixed freezing in.With for the polyclonal of CD31 Antibody dyes the slice of freezing.By the microvessel density in 20 high powers (200X) microscopic field, quantitative blood vessel life At.

Can be as mentioned previously, hemoglobin quantitation content (Schnaper, et al.,J. Cell Physiol. 1993, 256:235-246;Montesano, et al.,J. Cell Biol. 1983, 97:1648-1652; Stefansson, et al.,J. Biol. Chem. 2000, 276:8135-8141；And Gigli, et al.,J. Immunol. 1986, 100:1154-1164).It is rapidly frozen matrigel implantation material on dry ice, and is lyophilized overnight.It will dry Implantation material be resuspended in 0.4 mL, 1.0% saponin(es (Calbiochem) 1 hour, and be crushed by violent imbibition.? 14,000 X g centrifugations products 15 minutes, to remove any particle.Then by 405 nm measure absorbance, and with purifying The normal concentration of hemoglobin compares, and directly measures the hemoglobin concentration in supernatant.

The method for measuring cell migration

Cell migration assay has been described in the literature, for example, Brooks, et al.,J. Clin. Invest 1997, 99:1390-1398, the method for measuring cell migration are known to the skilled in the art.In a kind of survey as described herein In the method for measuring cell migration, migrated from transparent apertures (Transwell) with substrate (herein, CD105 and/or VEGF) coating The film of room washs transparent apertures, and BSA is used in combination to close nonspecific binding site.It is thin to harvest the tumour from subconfluent culture Born of the same parents, washing, and be resuspended in migration buffer solution in the presence of being with or without experiment antibody.Allowing tumor cell migration extremely After the downside of coated transparent pore membrane, removal remains in the cell on the upside of film, is used in combination crystal violet that will migrate to the cell of downside Dyeing.Then pass through the direct cell count of each microscopic field, quantitative cell migration.

The method for measuring cell Proliferation

By methods known to those skilled in the art, cell Proliferation can be measured.It can be by the people of subconfluent as described herein Endothelial cell (HUVEC) is resuspended in the proliferation buffer solution containing low (5.0%) serum and (comes from ECV or ECVL being with or without In the presence of the CM (25 μ L) of cell) in, and allow endothelial cell proliferation 24 hours.It is tried by using commercially available WST-1 It tests kit (Chemicon) and measures mitochondrial dehydrogenase enzymatic activity, can quantify and be proliferated.In addition, being surveyed by using standard method Amount³H incorporations (She et al.,Int. J. Cancer, 108:251-257 (2004)), proliferation as described herein can be quantified.

It is known in the art to assess the other methods of cell Proliferation, and is considered herein.In embodiment in more detail Ground describes other non-limiting examples.

The method for inducing CDC, ADCC and opsonification

Various therapies have been directed to innate immunity of the enhancing body to the cell of conversion.Conventional effector method includes mending The cell dissolution (" CDC ") of body dependence, the cytotoxicity (" ADCC ") of the cell of antibody dependent and phagocytosis are (immune Globulin is coated with after target cell, and reticuloendothelial system is purged).It is known that in the presence of antibody, certain effector cells (lymphoid cell of receptor such as combined with surface, antibody Fc district) mediates the antibody dependent for being directed to target cell The cytotoxicity (" ADCC ") of cell is reacted.By means of ADCC, these effector cells play for the thin of such target cell Cellular lysis activity.

Two class ADCC reactions have been confirmed in vitro.In classical ADCC reactions, effector cell is attached to antibody packet On the target cell of quilt, then cause target cell cell dissolution (A. H. Greenberg et al., “Characteristics Of The Effector Cells Mediating Cytotoxicity Against Antibody-Coated Target Cells,” Immunology, page 21,719 (1975)).Effector cell and target are thin This combination between born of the same parents, the phase interaction being originated between the areas Fc of antibody and the Fc receptors of effector cell of coating target cell With.One of this kind of ADCC reaction the disadvantage is that, it can be hindered by the antigen-antibody complex recycled, the compound warp It is often related with various diseases, Fc receptors (the I. C. M. for the antibody competition effector cell that they are combined with target cell MacLennan, “Competition For Receptors For Immunoglobulin On Cytotoxic Lymphocytes,” Clin. Exp. Immunol, page 10,275 (1972)).This due to classical ADCC lacks Point can use the second class ADCC to react, i.e. the ADCC of antibody guiding.In the ADCC of antibody guiding, make target special first The antibody of property is combined with effector cell, the compound then made pass through antibody " direction " on targeted cell surface it Specific antigen.Advantageously, the ADCC of antibody guiding may not be deposited by the antigen-antibody complex recycled in host system Influence.It is usually weaker by the areas Fc/interaction between the Fc receptors antibody being implemented in combination with and effector cell.Also, In some cases, antibody will not keep being combined the period for being enough to allow target cell to crack with effector cell.In view of this Potential problem is pre-processed using polyethylene glycol and phthalic acid ester oil mixture, makes antibody combination effector cell (J. F. Jones and D. M. Segal,J. Immunol, 125, the 926-33 pages (1980)).But in antibody-effect The toxic effect to body for answering any polyethylene glycol and phthalic acid the ester oil residue in cell complexes that may have, can Applicability of this method for interior therapeutic can be reduced.

Or, it has been proposed that enhance the ADCC of antibody guiding by using the adjuvant chemotherapy of cytotoxic drug Method (I. R. Mackay et al.,Cancer Immunol. Immunother, 16, the 98-100 pages (1983)).With It is well-known in the art in testing the experiment of ADCC, for example, U.S. Patent number 5,756,097.

Therefore, present embodiment provides such antibody：The antibody can be in conjunction in new vessels formation or blood vessel The cell or the antibody that work in generation can enhance the phagocytosis and kill of the cell, to be protected in reinforcement Shield.Other antibody and its function fragment with same effect are also provided, they specifically combine or preferentially combine in this way The combinable binding site of antibody or epitope, or immune response can occur with it.

The antibody can also for new vessels formed or angiogenesis in the cell that works (for example, endothelium is thin Born of the same parents) it is opsonic, or show conditioner activity.It will be appreciated by those skilled in the art that " conditioner activity " indicates conditioning Following abilities of element (being typically antibody or serum factor C3b)：The opsonin combination antigen or cell receptor, to promote antigen Or the combination of cell receptor and phagocyte, and thereby enhance phagocytosis.When being coated with by opsonin antibody, certain cells become Have a great attraction to phagocyte (such as neutrophil cell and macrophage), and they are from the clearance rate in blood flow It dramatically increases.With for example in U.S. Patent number 6, any usual manner described in 610,293 can measure conditioner activity.

In another non-limiting embodiment, the trouble of the illness with new vessels illness or angiogenesis-dependent Person is from angiogenesis released antigen/peptide (for example, CD105).These antigen/peptides can be " tumor associated antigen ".This can be given The antibody for the antigen/peptide (for example, CD105) is applied to the entire patient of sample, and can be started as described herein any Approach, to induce CDC, ADCC, opsonification or the cell-mediated kill in the form of any other.

Other experiments

Other experiments known in the art can be used for testing preparation described herein (such as, for example, in the following embodiments Description those of) effect.

Specific implementation mode

With reference to following non-limiting embodiment, the application may be better understood, the embodiment is as the application's Exemplary implementation scheme and provide.Following embodiments are provided, so that more fully illustrative example embodiment is not answered still It is construed to the wide scope of limitation the application.Although certain embodiments of the application have been shown and described herein, show And be clear to, such embodiment is only provided as embodiment.Many variations can be made, changes and replaces；It should be appreciated that Different change of the embodiment described herein can be used for putting into practice method described herein.

Embodiment 1:The exemplary dose timetable of application

The optimal dosage timetable of the application of antibody and its antigen-binding fragment can use art-recognized and as described above Method determines.

In one non-limiting embodiment, antibody described herein can be applied to subject through each period. Can following administration of antibodies or its antigen-binding fragment one or more dosage：2 times a week, 1 times a week, 1 time every 2 weeks, every 3 It is 1 time all, 4 weeks 1 time every, 6 weeks 1 time every, 8 weeks 1 time every, 12 weeks 1 time or any Zhou Zuhe between them every.Administration is also contemplated for follow Ring, such as, for example, 1 times a week or 2 administration of antibodies, it is for 4 weeks, it does not treat then 2 weeks.It is contemplated herein that additional gives Medicine recycles, including for example：Dosage described herein and the various combination recycled weekly.

Embodiment 2:BIAcore (surface plasma body resonant vibrations:SPR it) analyzes

It is analyzed using such as BIAcore, uses standard scheme, it can be estimated that the affinity of antibody.In brief, by anti-group of ammonia On acidity scale label antibody coupling to BIAcore chips, the BIAcore chips are used to capture the recombined human CD105 of His- labels, weight Group people CD105 is used to measure the combination of anti-CD105 antibody again.In minimum 2 chips prepare batch and 8 analysis batches, Carry out the exploitation of SPR experiments.In the exploitation of the experiment, parameters described below is assessed：

(a) coupling of anti-his antibody and CM5 chips

Anti- his tag antibodies are coupled on BIAcore CM5 chips using EDC/NHS by conventional amine chemical method.It will Optimize reaction condition (concentration and pH).

(b) regeneration of the combination of people VEGF and biologic sensor chip

Using various buffers (based on pervious experience) come the antibody of elution of bound, tester VEGF is combined and regeneration chip Condition.Once having developed for regenerated alternative approach, the interior combination for measuring one single chip surface is recycled at least 25 Capacity and background.Target is to obtain<10 RU/ cycle average background increase and<The average size of 1%/cycle reduces.

(c) combination of people CD105

The dose response for measuring people CD105, to determine the concentration being suitble to close to maximum combined.

(d) combination of anti-CD105 antibody

The dose response for measuring anti-CD105 antibody, to determine that (it may include pair for dynamic experiment or balance Binding experiment Than relative dynamics constant k_aAnd k_d, or pass through parallel line method compare relative potency) OK range.

(e) experiment before verification

Using different chips, different flow cell and in varied situations, repeatedly Binding experiment at least 3 under selected conditions It is secondary, to obtain the accuracy about measurement and the preliminary information of accuracy.It is carried out in HBS-EP running buffers at 25 DEG C All BIAcore experiments.

Embodiment 3:The ELISA combined for anti-CD105 antibody

ELISA can be used for measuring the combination of anti-CD105 antibody and CD105.In brief, it according to following step, carries out ELISA：

1. with 100 holes μ l/, with MAB9811-01 (the anti-CD105 antibody of monoclonal, in PBS, 1500 ng/ml) coating Plate.It is incubated overnight (16-24 hours) with sealer cover plate, and at 4 DEG C.

2. washing plate 2 times with 200 μ l PBS (not having tween).

3. washing plate 3 times with the PBS (PBS-T) containing tween.

4. adding the CD105 of 100 ng/ml in the PBS-T with 0.1% BSA in 100 holes μ l/, and in incubation at room temperature 60 Minute.

5. washing plate 3 times with PBS-T.

5. in experimental port：Be added 100 holes μ l/ in 20,10,4,2,1,0.5 and 0.2 ng/ml (containing 0.1% BSA PBS-T in dilute) anti-CD105 antibody, and incubation at room temperature 60 minutes.In negative control hole：100 holes μ l/ are added The matched control antibodies of isotype.

7. washing plate 3 times with PBS-T.

8. the goat anti-human IgG being conjugated with HRP in 100 holes μ l/ is added into all holes (in the PBS-T containing 0.1% BSA In 1:10000 dilutions)；In incubation at room temperature 30-60 minutes.

9. washing plate 5 times with PBS-T.

10. the tmb substrate solution in 100 holes μ l/ is added, and in the dark naked it is incubated 15 minutes.

11. by the way that the TMB stop baths in 100 holes μ l/ are added, reaction is terminated.

Run sample in triplicate, and optical density is read, to build standard curve, and measure binding constant.It uses StudentShi t- are examined or another standard test, for statistical analysis.

It should be appreciated that test antibody and the combination of VEGF can be come using similar scheme.

Embodiment 4:Antibody affinity and expression CD105 cell on available epitope number

Analyzed using scatchard plot, use standard scheme, it can be estimated that antibody affinity and expression CD105 cell on The number of available epitope.

In brief, the KM-3 leukaemia cell and Ya of radiolabeled anti-CD105 antibody and expression CD105 are carried out The scatchard plot analysis of HUVEC in the proliferation converged bound directly.Using Iodo-Gen, and according to art technology Standard method known to personnel is used¹²⁵I individually radioactively marks the anti-CD105 antibody of purifying.About each IgG molecules In average iodine atomicity, measure radiolabeled anti-CD105 antibody respectively.Use each of fixed amount (0.1 μ g)¹²⁵KM-3 the or HUVEC cells of the expression CD105 of mAb and 2 times of series of incremental of I- labels, carry out titration experiments, anti-to measure Original combines activity.According to known methods, it is combined the scatchard plot analysis of data.Pass through the analysis, estimation balance The average maximum number of constant and the mAb of each cell combination.

Embodiment 5:The Western blot of blocking activity

By western blot, the activation for blocking CD105 stimulations, expression CD105 cell of anti-CD105 antibody can be tested Ability, with detection participate in CD105 signal transduction paths albumen phosphorylation.

According to known western blotting technique, carry out western blot analysis, with identify phosphorylation Smad1/5/8 or Smad2.Identify to PSmad1 and PSmad2 antibody specificities the Smad1/5 or phosphoric acid of the phosphorylation in the endothelial cell of untransfected The Smad2 of change.Using the primary antibody for Smad1, Smad2, Smad5, Id1 (Santa Cruz) and CD105, sample is detected In molecule.By the chemiluminescence (ECL) of enhancing, it is detected.

Embodiment 6:HUVEC growth inhibition and³H- thymidine incorporations are tested

Many experiments can be used for assessing the inhibition of cell growth.

In one embodiment, in CO₂In incubator, at 37 DEG C, under conditions of subconfluent, in 75-cm²Flask HUVEC is cultivated in (Falcon, Becton-Dickinson, Franklin Lakes, NJ).By molten with Hanks Balanced Salt Liquid (containing 15 mM EDTA, in 25 mM HEPES buffer solutions pH 7.3) is incubated 15 min at 37 DEG C, makes cell detachment.With After ice-cold PBS is washed 2 times, with 25, cell is resuspended in Endothelial Cell Growth Medium by the concentration of 000 cell/ml In.

In other experiments, in the Endothelial Cell Growth Medium without containing FBS and Medulla Bovis seu Bubali extract, suspends and cultivate Human umbilical vein endothelial cells (HUVEC).200 μ l aliquots of cell suspending liquid are seeded to each hole of 96- well culture plates In.In CO₂Cell pellet overnight is cultivated in incubator at 37 DEG C, it is anti-that anti-CD105 antibody, anti-VEGF are then added in triplicate Body, the combination of anti-CD105 antibody and anti-VEGF antibodies, control IgG or TGF-β 1.Culture plate is kept 72 in the incubator Hour, in the meantime, replace fresh culture and antibody or control within every 24 hours.It will³Each hole is added in H- thymidines (1 μ Ci) In, plate is incubated 20 hours.Cell is washed with PBS, then with trypsase-EDTA (0.05% tryptoses in 100 holes μ l/ Enzyme, 0.53 mM EDTA) 15 min are handled at 37 DEG C.It, will be thin using Harvester 96 (TOMTEC, Hamden, CT) In born of the same parents' harvest to glass fiber filter (Wallac Printed FiltermatA), and in 1540 MicroBeta of Trilux It is measured in liquid scintillation and luminescent counter (Wallac, Turku, Finland)³H- radioactivity.

Embodiment 7:Cellular migration inhibition is tested

Migration (chemokinesis), the measurement as cell Proliferation and activation are measured using Boyden chamber.

In brief, cell migration is assessed as follows：At 4 DEG C Costar nucleopores filter paper (8 holes mm) is coated with fibronectin Overnight.The room is washed with phosphate buffered saline (PBS) (PBS), under being filled with DMEM (being with or without serum, be with or without TGF-β 3) Room.Exist with trypsin treatment cell, and in control antibodies, anti-CD105 antibody, anti-VEGF antibodies or combination thereof Under, with 50, the final concentration of 000 cell/ml is suspended in DMEM.Upper chamber is added in 150 μ l aliquots of cell suspending liquid In, and be incubated at 37 DEG C.After 16 hours, cell is washed, upper surface is dried, to remove the cell not migrated.In methyl alcohol Fixed film, is washed with water, and dyes, and counts the number of existing cell on the lower surface.

Embodiment 8:ADCC is tested

Use example scheme described as follows, can be about their natural killer (NK) cell for generating IL-2 activation and induction ADCC Ability, assess antibody as described herein.

NK is detached and the generation of the NK cells of IL-2 activation

PBMC is detached, and allows to stand 24 hours at 4 DEG C in the RPMI containing 10% FBS.Then PBMC is put into containing 2% In the RPMI of FBS (total volume=50 mL), by 10mL cell suspending liquids in culture dish bed board.It is small that PBMC 2 is incubated at 37 DEG C When, collect not adherent cell.With 8 X 10⁶/ mL cultivates NK cells 48 hours, then normal training with the IL-2 of 1000 U/mL It supports 5-8 days, is subsequently used in experiment.

Natural cytotoxicity and ADCC experiments

NK cells are scraped from culture, and are collected in 50mL conical pipes.Cell is washed with RPMI complete mediums 1 time, and It is centrifuged 10 minutes in 1200 rpm.Then NK cells are resuspended in 5mL RPMI complete mediums, and counted.Carry out Before experiment, NK cell counts are standardized as 10:1 effector:The ratio between target.By standardized NK plating cells, and will The anti-CD105 antibody of 10 μ L is added in specified hole, is incubated 30 minutes at 37 DEG C.Control sample includes untreated or control The cell colony of antibody processing.

Purpose target cell (HUVEC cells) is collected, washing centrifuges 10 minutes in 1200 rpm, and is resuspended in 5 In mL RPMI complete mediums.Target cell is washed again, and is resuspended in the RPMI of serum-free, until 1 x 10⁶Carefully The final concentration of born of the same parents/mL.Then target cell is marked at 37 DEG C with the Calcein-Safranine T of final concentration of 5 μ g/mL 1 hour, then It is washed 2 times with RPMI complete mediums.Then suspend target cell again, and is added in NK cell holes.Target is incubated at 37 DEG C Cell/NK cells combine 4 hours.After incubation, centrifuges plate 5 minutes in 1200 rpm, wash cell, and be resuspended in DPBS In.Using the excitation/emission of 450/530 nm, fluorescence is read, the transmitting is the measurement killed by antibody-mediated cell.

Embodiment 9:The estimation of stability of TRC105 (anti-CD105 antibody) preparation

The present inventor identifies the preparation that sufficiently stable property is provided for the concentration higher than 5 mg/ml TRC105.Preliminary research is aobvious Show, TRC105 can be concentrated at least 25 mg/ml, without any apparent solubility problem.Therefore, 25 mg/ml are selected TRC105 is as the initial concentration for screening study.In these flow of research, assessment is up to the system of 50 mg/ml TRC105 Agent.The also additional preparation of concentration of the assessment with 7 mg/ml TRC105 and 100 mg/ml TRC105.

Program

Ultraviolet (UV) absorption spectrum.

The albumen concentration in various stability samples is measured using UV spectrum.Bulk materials (7.0 mg/mL) are 280 The absorbance of nm is measured as 1.13 using 1 mm light paths room, leads to the extinction coefficient of 1.61 mL/mg*cm.The value is used for the project In all calculating.It further measures and is carried out using 0.0096 rooms cm more suitable for enriched sample.

The incubation of TRC105 thermal stability samples.

Each thermal stability preparation is by locking 0.22 micron of Millex filter of syringe attachment, then decile with Luer It is dispensed into 1 mL freeze-drying bottles.Each bottle is sealed with butyl rubber stoppers and with aluminium cap crimping.By sample in assigned temperature Under be placed on stability chamber, and taken out after shown incubation period within several weeks.

The freeze thawing and stirring of TRC105.

Freeze thawing (F/T) sample is freezed 25 minutes, 25 minutes or zero degree, 3 times or 5 times are then melted.All freezings are equal It is carried out at -80 DEG C.

It is checked before returning to refrigerator and melts whether sample melts completely.Each freeze thawing sample contains 100 μ L solution and storage There are in 0.5 mL Eppendorf pipes.Each sample contains 0.1% F68,0.005% Tween 80 or is free of surface-active Agent.

Stirring study sample is prepared as every bottle of 350 mL, and labeled as static or shake.All samples are transferred to 4 DEG C In cold house.It will be placed on the orbital shaker operated with 150 RPM labeled as the sample of shake.Static sample is placed on closely rail On the specimen holder of road shaking table.Each sample contains 0.1% F68,0.005% Tween 80 or is free of surfactant.By sample It is incubated 24 hours in shown condition.

Size exclusion chromatography method (SEC).

The mobile phase containing 0.2 M sodium dihydrogen phosphates is prepared, and pH7.0 is adjusted to using 1.0 M sodium hydroxides.TSK Gel G3000 PWxl columns (7.8 mm x 30 cm, 7 μm of particle # P0047-02PN) are used for all separation.Use 1 mL/ The flow velocity of min, and Detection wavelength is set as 280 nm.Volume injected is 2 μ L for 25 mg/mL samples, and for 50 Mg/mL samples are 1 μ L.Buffer blank is run before each triplicate sample analysis, and these chromatographies are suitably subtracted It removes.All peaks are incorporated into Chromeleon 6.08 and carry out.

Capillary IEF (cIEF)。

PA 800 of the electricity such as capillary (cIEF) analysis as published by Beckman CoulterplusDescribed in application guide into Row.Describe in more detail visible Mack et al. (Electrophoresis 2009, 30(23), 4049-4058).All points Analysis in environment temperature using operating with the neutral capillary (50 μm of internal diameters) of 30 cm total lengths (20 cm effective lengths) Beckman Coulter P/ACE MDQ systems (Beckman Coulter, Inc.;Brea, CA) it carries out.Neutral hair Tubule by using Gao et al. (Anal Chem 2004, 76(24), 7179-7186) described in method by poly- (acryloyl Amine) capillary wall is fixed to prepare.All cIEF samples are by mixing the destination protein of 0.25 mg/mL and containing two Property electrolyte, cathode stabilizers, anodic stabilization agent and pI labels the mixtures of 3M urea-cIEF gels prepare.By sample Continue 4.1 min with 9.5 psi pressure injections to capillary, passes through it in anodolyte and catholyte after the time Apply 15 min of voltage of 25 kV between matter and is focused.After the step, between anodolyte and chemical fixatives 30 min are fixed with 30 kV chemistry.PI is marked and destination protein carries out during fixing step used in the absorbance of 280 nm Detection.The pI of albumen is from pI vsThe gained regression equation of the first peak time obtained from pI standard items calculates.

As a result it and discusses

Research 1

The sample of TRC105 is successfully concentrated into the concentration higher than 5 mg/mL.As a result, research 1 uses 25 mg/mL's Albumen concentration carries out.Sample is stored two weeks at 40 DEG C.15 kinds of preparations are prepared, the shadow of pH and buffer solution composition are focused on It rings (table 2).The primary analysis method of stability for assessing these samples is SEC.After being stored two weeks at 40 DEG C, pass through SEC sees the very small variation (table 3) of the amount of content of monomer or high molecular weight aggregates.All formulations still contain after storage More than 99% monomer.

The composition for the TRC105 preparations assessed in the research of table 2. 1

The monomer and aggregation that by SEC are measured of the table 3. in preparation of initial and two weeks (t2) time points from research 1 Horizontal summary

It was found that TRC105 is highly stable.

Research 2

Result from research 1 devises recent studies in hand.In research 2, ranging from 4.0 to 5.5 pH and second are had evaluated Hydrochlorate, histidine and citrate.Tension regulator/stabilizer of selection is D-sorbite and trehalose.Finally, in addition to 25 Other than the preparation of mg/ml, three kinds of preparations of 50 mg/ml are also checked for.

The composition for the TRC105 preparations assessed in the research of table 4. 2

In view of the stability of TRC105, these samples are stored into surrounding at 40 DEG C, to attempt more to distinguish the system Agent.Initial screening tool is SEC.

The monomer that by SEC is measured and aggregation water of the table 5. in preparation of the initial and 4th week time point from research 1 Flat summary

Similar with seeing in research 1, the content of monomer of nearly all preparation is above 99.5%, or even at 40 DEG C 1 After month (table 5).One sample seems to show increased aggregation level, this is F16；This is from the point of view of not retesting the sample No is legitimate reading.This shows that pH 4 seems to provide enough and comparable stability in the acetate of pH 5.5 in histidine.Mirror In the concern of citrate and stimulation, it is removed from further consideration.

These samples are also analyzed using capillary isoelectric focusing (CIEF).The integral central of cIEF electrophoretograms is calculated For the first moment.These values are considered the average pI values (table 6) of sample.

Average pI value of the table 6. in TRC preparation of the initial and surrounding time point from research 2

In short, the variation of the pI values of any preparation is small, show deamidation, that is, form the Asn side chains of electrification Asp products Hydrolysis, relatively slowly (table 6).Therefore, these preparations of TRC105 seemingly (that is, the little or no aggregation) of physically stable and Chemically stable (that is, without fragmentation and little or no deamidation).

Other concerns are that whether these higher concentration preparations show bad viscosity, thus limit its clinical application.

The dynamic viscosity of the TRC105 in the acetate of ~ 25 and ~ 50 mg/ml and histidine buffer of table 7.

Even show the viscosity less than 2 cSt in 50 mg/ml, TRC105, far below causing for limitation processing and Any value of the worry of application (referring to table 7).

Stirring research

Although heat stress can be used for distinguishing the preparation of different stability, it is not uniquely stress of can encountering of albumen.Albumen Interfacial failure is common, and must assess to interface stress sensibility.This using stir and repeat freeze thawing (F/T) recycle into Row.

Table 8. stirs 24 hours TRC105 preparations with 150 rpm and matched static sample is contained by the monomer of SEC Amount.All samples are held in 4 DEG C

Monomer does not have significantly sacrificing, wherein all formulations to show to compare higher than 99.7% monomer and with static state after stirring It is not significantly different (table 8).Add surfactant, such as Pluronic F-68 or polysorbate80, it appears that do not help or Stability after injury stirring.

F/T is studied

Check TRC105 to interface stress sensibility Section 2 research involve an exposure to repeatedly Frozen-thawed cycled.This is not only provided About the information of interface (as ice-water interface) damage, it is additionally provided about whether TRC105 samples can freeze for subsequent Analysis or the essential information freezed during transport.

Table 9. is subjected to the content of monomer by SEC of the TRC105 preparations of 0,3 and 5 F/T cycles.

Content of monomer has almost no change (table 9) after repeating F/T cycles., it is surprising that addition surfactant, such as Pluronic F-68 or polysorbate80 do not change the stability after repetition F/T cycles.Together with stirring data, without mark As showing that TRC105 has surface-active in the degree of worry interface damage.Therefore, surfactant need not be used for liquid system Agent.

It summarizes

Preparation screening research is carried out to TRC105 in the up to albumen concentration of 50 mg/ml.This albumen is quite stable, is shown Showing is reduced by the only a small amount of of content of monomer of SEC, or even at 40 DEG C after storage four weeks.It is not seen and is taken office completely by SEC What fragmentation.Meanwhile cIEF analysis shows that, observe whole pI only small variation, show discussion pH ranges (4.0 to 5.5) deamidation is minimum.Not for the evidence of the sensibility of interface damage, show not needing in the liquid preparation of TRC105 Surfactant.

Most important preparation is using the acetate buffer of pH 4.0 or the histidine buffer of pH 5.5, together with sorbose Alcohol or trehalose are as tension regulator and stabilizer.

Embodiment 10:The long-time stability of TRC105 preparations are evaluated

Evaluation steady in a long-term is carried out with the result in checking research 1 and 2.The formulation samples presented in table 10 are stored in 5 DEG C and 25 ℃。

Table 10：Preparation for checking research

Preparation is numbered	Preparation	[albumen]
			F01	5.5,20 mM histidines of pH, 240 mM trehaloses	25 mg/ml
F02	5.5,20 mM histidines of pH, 240 mM D-sorbites	25 mg/ml
			F03	4.0,20 mM acetates of pH, 240 mM D-sorbites	25 mg/ml
F04	5.5,20 mM histidines of pH, 240 mM trehaloses	50 mg/ml
			F05	4.0,20 mM acetates of pH, 240 mM trehaloses	50 mg/ml
F06	5.5,20 mM histidines of pH, 240 mM trehaloses	100 mg/ml
			F07	4.0,20 mM acetates of pH, 240 mM trehaloses	100 mg/ml

As a result it and discusses

Seven kinds of test formulations assess 1 year process at 5 DEG C, and 6 months processes are assessed at 25 DEG C.At 5 DEG C, all formulations all tables It is now good.The visual appearance at all samples all time points in this study is clarification and colourless.In general, all novel formulations PH value is kept constant in the duration of the research (referring to table 11 and 12).Value 0.1 to 0.2 unit more slightly higher than target, but protect It is fixed to keep steady.No matter concentration how, by UV measure novel formulation concentration value desired value ~ 3-4 % in keep constant (referring to table 11 and 12).These values are in the course of the research without significant changes.It is minimum (referring to table 13) by SEC loss of monomer at 5 DEG C, and And it is minimum (referring to table 15 and 16) by cIEF chemical degradations amount.Compared with the result of reference standard product, although CD105 is combined ELISA results have some to change (in addition to the initial results of F05), but all values receive 50% to 150% within standard, This is consistent (referring to table 17) with the ELISA colour developing stages measured.CE-SDS test the result shows that, at 5 DEG C more than 1 year In the process, the sample is suitable with reference material (data are not shown).

Although being stored in 5 DEG C of sample to be basically unchanged, formulation parameters have influence really on the stability of 25 DEG C of samples. Most notably, the preparation (F03, F05, F07) containing acetate buffer system is shown than containing histidine buffer The larger reduction of the percentage monomer of the preparation (F01, F02, F04 and F06) of agent (referring to table 14).Meanwhile albumen concentration changes Change seems there is very little or none influence.50 mg/mL counterparts of two kind of 100 mg/mL preparations (F06, F07) and they (F04, F05) is suitable in terms of their SEC overviews and cIEF overviews.

PH of the table 11. at 5 DEG C and the UV concentration in terms of mg/mL

PH of the table 12. at 25 DEG C and the UV concentration in terms of mg/mL

Size exclusion chromatography (SEC) of the table 13. at 5 DEG C

Size exclusion chromatography (SEC) of the table 14. at 25 DEG C

Table 15：It is averaged pI in 5 DEG C of cIEF-

Table 16：It is averaged pI in 25 DEG C of cIEF-

Table 17：In 5 DEG C of CD105 combinations ELISA

Embodiment 11:The long-time stability of 4 mg/mL TRC105 preparations are evaluated

To (being criticized with the TRC105 that 4 mg/mL are prepared in the 17 mM phosphate buffers with 145 mM sodium chloride (pH 7.2) Number FIN-0536) carry out stability study.36 months of stability data are recommending (2-8 DEG C) of condition of storage available, And acceleration environment of one month of data in 25 DEG C/60%RH is available.Stability data is presented in following table.For appointing for progress Significant changes are not observed in what test.Therefore, these are studies have shown that when being stored in 2-8 DEG C, with 145 mM chlorinations Stablized at least 36 months with the TRC105 that 4 mg/mL are prepared in 17 mM phosphate buffers of sodium (pH 7.2).

Table 18：In the stability data of the TRC105 of 2-8 DEG C of recommendation storage conditions

Test	Initially	6 months	9 months	12 months
					Appearance and description	Clear colorless solution.Light grey Dust sample particle.	Clear colorless solution, and there is no appoint What visible granular.	Clear colorless solution, and exist slight White particles.	Clear colorless solution, and exist slight white Color particle.
Irreducibility SDS-PAGE	It is suitable with reference	It is suitable with reference	It is suitable with reference	It is suitable with reference
					Reproducibility SDS-PAGE	98%	96%	97%	98%
IEF	It is suitable with reference	It is suitable with reference	It is suitable with reference	It is suitable with reference
					SEC-HPLC	96.62%	96.97 %	96.91 %	96.77 %
It is combined by the CD105 of ELISA	It is relative to reference in conjunction with activity The 88% of standard items	It is relative to reference standard in conjunction with activity The 110% of product	It is relative to reference to mark in conjunction with activity The 104% of quasi- product	It is relative to reference standard in conjunction with activity The 91% of product
					UV absorbances (A₂₈₀)	4.3 mg/mL	4.2 mg/mL	4.2 mg/mL	4.2 mg/mL
pH	7.2	7.3	7.2	7.2
					Aseptic	It does not test	It does not test	It does not test	It does not test

Table 18 is continuous

Test	18 months	24 months	30 months	36 months
					Appearance and description	Clear colorless solution, and exist light Micro white particle.	Clear colorless solution, and there is no appoint What particle.	Clear colorless solution, and there is no appoint What particle.	Clear colorless solution, and there is no any Particle.
Irreducibility SDS-PAGE	It is suitable with reference	It is suitable with reference	It is suitable with reference	It is suitable with reference
					Reproducibility SDS-PAGE	97%	96%	97%	96%
IEF	It is suitable with reference	It is suitable with reference	It is suitable with reference	It is suitable with reference
					SEC-HPLC	96.32 %	96.86 %	96.60 %	94.04%
It is combined by the CD105 of ELISA	It is relative to reference in conjunction with activity The 88% of standard items	It is relative to reference standard in conjunction with activity The 136% of product	It is relative to reference to mark in conjunction with activity The 88% of quasi- product	It is relative to reference standard in conjunction with activity The 105% of product
					UV absorbances (A₂₈₀)	4.2 mg/mL	4.2 mg/mL	4.2 mg/mL	4.2 mg/mL
pH	7.2	7.2	7.2	7.3
					Aseptic	It does not test	Pass through	It does not test	Pass through

Table 19：In the stability data of the lot number FIN-0536 of the accelerating storage condition storage of 25 DEG C/60% RH

Test	Initially	1 month
			Appearance and description	Clear colorless solution.Light grey dust sample particle.	Clear colorless solution, and any visible granular is not present.
Irreducibility SDS-PAGE	It is suitable with reference	It is suitable with reference
			Reproducibility SDS-PAGE	98%	96%
IEF	It is suitable with reference	It is suitable with reference
			SEC-HPLC	96.62%	96.45%
It is combined by the CD105 of ELISA	It is 88% relative to reference standard product in conjunction with activity	It is 91% relative to reference standard product in conjunction with activity
			UV absorbances (A₂₈₀)	4.3 mg/mL	4.2 mg/mL
pH	7.2	7.3

The analysis program of SEC-HPLC and UV and the program described in embodiment 9 are identical.CD105 combinations ELISA's It is identical to analyze program and the program described in embodiment 3.Irreducibility SDS-PAGE, reproducibility SDS-PAGE and IEF It is as follows to analyze program description.

The SDS-PAGE of irreducibility albumen and peptide, coomassie dyeing

Identity and purity are assessed using Polyacrylamide Gel Electrophoresis.Albumen detaches under Denaturing.Using pre- The gel containing 4-12% Bis-Tris SDS of system.SDS is anionic detergent, negative to form band with protein-interacting The complex compound of electricity.These complex compounds migrate across polyacrylamide gel according to their size so that bigger compared with little albumen Albumen migrates faster.Standard items, control and test article are denaturalized and are loaded on gel.After completing operation, gel is with examining horse This indigo plant dyeing.Gel is scanned using image density meter.

The relative quantity of each colored zone in each test article road is determined by densitometry.This tittle is represented as pair In the percentage for total ribbon amount that the road obtains.Molecular weight (kDa) value of test article band based on the comparison with molecular weight marker come It determines.

The SDS-PAGE of reproducibility albumen, coomassie dyeing

SDS-PAGE methods for going back raw sample are (above) for identical described in non-reducing sample with 1.3.2 sections, wherein increasing Add reduction step (crack intermolecular and intramolecular disulfide bond to discharge protein protomer and other aggregations).In order to restore egg In vain, dithiothreitol (DTT) (50 mM) is added to sample and reference, is then heated five minutes at 85 DEG C.When operation restores albumen, Antioxidant is added to running buffer.

Horizontal isoelectric focusing (IEF)

Using the different charges in the case where applying electric power on albumen come protein isolate.PH gradient is established between a cathode and an anode, The pH value of middle cathode is higher.Since albumen has both sexes characteristic, they by with less than its pI positively charged value and be higher than it The electronegative value of pI.Under the influence of electric power, pH gradient is established and albumen will migrate and concentrate on their isoelectric point. Described program is related to using the isoelectric focusing of protein sample using the precast gel of horizontal versions.After electrophoresis, by gel coomassie Dyeing.The area of each band in each sample road is determined by densitometry.The area of band be calculated as always with area hundred Divide ratio.Also measure pI the and Rf values of each band.

Embodiment 12:The clinical test of colorectal cancer

This embodiment describes randomization, set blind, placebo, polycentric, the II phases and study, the research is used for The entry evaluation of the safety and effect of anti-CD105 antibody in colorectal cancer patients is provided.Recruited substantially about 100 to About 800 patients distribute about 50 to about 400 patients to treatment group, and about 50 to about 400 patients are distributed to placebo Group.The experiment will be by every two weeks using the anti-CD105 antibody or comfort of about 0.1 to about 10 mg/kg of intravenous repeated doses Agent composition continues 6-10 period.Chemotherapy can be used in all groups.VEGF inhibitor all can be used in all groups.Estimate The search time range of survey is about 6 months to about 5 years, and the follow-up therapy of respondent is such as shown at the end of Primary Study.Additionally Outcome measure it is as follows：

Main result is measured：Total response rate.One goal in research is proved with anti-CD105 Antybody therapies compared to control IgG Total response rate increase.

The secondary outcome measure that can be assessed includes：It is response duration time, the tumour progression time, total survival rate, serious Not serious adverse events.For example, treatment can prevent the progress (that is, stagnation) of disease, or can cause to improve.Alternatively It or extraly, can be about following one or more, measurement other purposes：Tumor load reduces, new vessels form reduction, Side effect reduction, adverse reaction reduction and/or patient compliance increase.

Embodiment 13:The clinical test of kidney

Present embodiment describes randomization, set blind, placebo, polycentric, the II phases and study, the research is through setting Entry evaluation of the meter for providing the safety sum of anti-CD105 antibody in the patient with clear-cell carcinoma (kidney). Substantially about 100 to about 800 patients have been recruited, about 50 to about 400 patients have been distributed to treatment group, it will be about 50 to about 400 Patient distributes to placebo.The experiment is made up of：Per 1-3 weeks, the intravenous anti-CD105 using repeated doses was anti- Body (about 0.1 to about 30 mg/kg) or placebo continue 3-6 cycle, or until progress.It can also make in 2 treatment groups Use interferon.VEGF inhibitor all can be used in Liang Ge treatment groups.The search time range of estimation is about 6 months to about 5 years, is answered The follow-up therapy for the person of answering is such as shown at the end of Primary Study.Additional outcome measure is as follows：

Main result is measured：Progresson free survival.One goal in research is after proving anti-CD105 Antybody therapies compared to comfort The increase of the progresson free survival of agent.

Embodiment 14:The clinical test of hepatocellular carcinoma

Present embodiment describes randomization, set blind, placebo, polycentric, the II phases and study, the research is through setting Entry evaluation of the meter for providing the safety sum of anti-CD105 antibody in the patient with hepatocellular carcinoma (liver cancer). Substantially about 100 to about 800 patients have been recruited, about 50 to about 400 patients have been distributed to treatment group, it will be about 50 to about 400 Patient distributes to placebo.VEGF inhibitor all can be used in two groups.The experiment is made up of：Per 1-3 weeks, vein Interior anti-CD105 antibody (about 0.1 to about 30 mg/kg) or placebo using repeated doses, or until progress.Estimation is ground It is about 6 months to about 5 years to study carefully time range, and the follow-up therapy of respondent is such as shown at the end of Primary Study.Additional result Measurement is as follows：

Embodiment 15:The clinical test of oophoroma

Present embodiment describes randomization, set blind, placebo, polycentric, the II phases and study, the research is through setting Entry evaluation of the meter for providing the safety sum of anti-CD105 antibody in the patient with oophoroma.It has recruited big About 100 to about 800 patients are caused, about 50 to about 400 patients are distributed to treatment group, it will about 50 to about 400 patient's distribution To placebo.The experiment is made up of：Per 1-3 weeks, the intravenous chimeric anti-CD105 antibody for applying repeated doses (about 0.1 to about 30 mg/kg) or placebo continue 5 cycles.Chemotherapy can also be used in Liang Ge treatment groups.Two VEGF inhibitor all can be used in treatment group.The search time range of estimation is about 6 months to about 5 years, the follow-up treatment of respondent Method is such as shown at the end of Primary Study.Additional outcome measure is as follows：

Main result is measured:Progresson free survival.One goal in research is after proving anti-CD105 Antybody therapies compared to comfort The increase of the progresson free survival of agent.

Embodiment 16:The treatment of age-related macular degeneration

First research

By the anti-CD105 antibody of intravitreal injection or control antibodies, treatment shows the trouble of age-related macular degeneration Person, to mitigate or prevent new vessels formation, the development of macular disease and retinal damage.

As the first step for the treatment of, patient receives full eye examination, to establish the baseline of eye health.Eye examination includes：Examine eye Mirror indirect review method, slit lamp biomicroscope's inspection, peripheral retina inspection, measure of intraocular pressure, visual acuity (it is independent and Most preferably correcting) symptomatology, fundus photography, fluorescein angiography, optical coherence tomography, retina electricity trace Art and a-scan measure.

After trial inspection, the impacted of AMD is shown by what intravitreal injection as described above gave patient Eye.If eyes are all impacted, them can be individually treated.Eye to be treated is injected with ophthalmic solution.

After treatment, in first (1) day, second (2) day, the 7th (7) day, the 15th (15) day, the 30th (30) day and 60 (60) days, and hereafter in every month, 2, check the eye of patient.Because of the possibility of recurrence, patient should every month thereafter It comes back for inspecting periodically.In each check day, the nature of glass liquefaction of patient is monitored.In addition, using the inspection that there is sclera to collapse Glasses indirect review method, the rear vitreous body for monitoring patient are detached from.Finally, it is taken the photograph by regular dynamic retinoscopy, optical coherence body layer Shadow art and fluorescein angiography picture continuously monitor the degree for the AMD that patient is presented, to monitor subretinal fluid, blood Liquid, exudate, the presence that RPE is detached from, capsule retina changes or the presence of celadon subretinal neovascularization.If Observe the sign that recurrence new vessels are formed, it may be desired to additional treatment.Additional treatment can weekly or monthly be given.? In one preferred embodiment, after first treatment, interval carries out successive treatment in 1-6 months.

Second research

Purpose：Confirm age-related macular degeneration (AMD) of the intravitreous anti-CD105 antibody for treating new vessels The effect of.VEGF inhibitor all can be used in all patients.

Method：The positions the 50-500 patient (50-500 of (CNV) is generated with the subfoveal choroidal neovascular from AMD Eye) it participates in studying in the site of approval.

The standard injected again is, in macula lutea there are liquid, central retina thickness (CRT) increase at least 100 microns, At least five letter vision loss related with increased liquid in macula lutea, the CNV of new type or new punctum luteum hemorrhage.Main knot Fruit measurement is to be less than the ratio of 15 alphabetical vision loss after 12 months.At 1 week, 1 month, then at 5-12 Every month in month, the visual acuity most preferably corrected measures and clinical eye examination.

It compares with baseline, measures average visual acuity and average CRT.Record eye and/or whole body side effect.

Embodiment 17:Humanization-removes immune antibody sequence

The immune anti-CD105 antibody that goes of the humanization of separation can include to have such as SEQ ID NO:Amino acid sequence shown in 11 Heavy chain variable region and with such as SEQ ID NO:The light chain variable region of amino acid sequence shown in 12.

Humanization-goes other non-limiting examples of immune heavy chain to include, but are not limited to SEQ ID NO:13,14,15 and 16。

Humanization-goes other non-limiting examples of immune light chain to include, but are not limited to SEQ ID NO:17,18,19, 20,21,22 and 23.

Can realize otherwise or other modes implement the embodiment described herein aspect, without departing from its essence God or essential characteristic.Therefore, illustration it is all from the aspect of present disclosure, and be not limiting, it is intended that wrap wherein Include all changes fallen into the meaning and scope of equivalent scheme.

Sequence table

<110> TRACON PHARMACEUTICALS, INC.

<120>Antibody preparation and application thereof

<130> 35882-714.601

<140>

<141>

<150> 61/697,111

<151> 2012-09-05

<160> 23

<170> PatentIn version 3.5

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His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

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Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

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Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

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Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

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Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

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Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

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Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

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Ala Glu Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

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Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr

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Thr Leu Thr Val Ser Ser

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Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

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Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

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Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

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Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

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Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

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Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

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Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

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Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

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Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

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Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

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Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

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Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

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Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

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Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

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Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

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Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Trp Arg Arg Phe Phe Asp Ser

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

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35 40 45

Gly Glu Ala Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

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Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 12

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<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

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35 40 45

Ala Glu Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Glu Ile Arg Ser Gln Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Glu Ile Arg Ser Arg Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 16

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Glu Ile Arg Ser Lys Ala Ser Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Trp Arg Arg Phe Phe Asp Ser Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 17

<211> 106

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<220>

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<400> 17

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 18

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<400> 18

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Ser Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 19

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<400> 19

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 20

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<400> 20

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Ser Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

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<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Ser Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 22

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<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Ser Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

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<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp Ala Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

Claims

2. the preparation of claim 1, wherein such as measured by size exclusion chromatography method (SEC), at least 95% anti-CD105 Antibody exists after being stored at about 2 to 8 DEG C at least 12 months as monomer.

3. the preparation of claim 1, wherein such as measured by size exclusion chromatography method (SEC), at least 95% anti-CD105 Antibody exists after storing at least six moon at about 25 DEG C as monomer.

4. the preparation of claim 1, wherein such as measured by size exclusion chromatography method (SEC), at least 90% anti-CD105 Antibody exists after storing at least six moon at about 25 DEG C as monomer.

5. the preparation of claim 1, wherein the buffer is histidine or phosphate buffered saline (PBS).

6. the preparation of claim 1, wherein the buffer is acetate, and pH is about 4.

7. the preparation of claim 1, wherein the buffer is histidine, and pH is about 5.5.

8. the preparation of claim 1, wherein the anti-CD105 antibody or its antigen-binding fragment store at least 12 at about 2 to 8 DEG C Display is combined by about the 50 to 150% of CD105 ELISA binding assays after a month.

9. the preparation of claim 1, wherein as measured by Capillary Electrophoresis-isoelectric focusing, at least 12 are stored at 2 to 8 DEG C After a month, the average isoelectric point (pI) of the anti-CD105 antibody is about 8.7 to about 9.2.

10. the preparation of claim 1, wherein the anti-CD105 antibody includes to have such as SEQ ID NO:Amino acid sequence shown in 1 Light chain variable region (V_L)；With such as SEQ ID NO:Constant region of light chain (the C of amino acid sequence shown in 2_L)；With such as SEQ ID NO:Heavy chain variable region (the V of amino acid sequence shown in 3_H)；With with such as SEQ ID NO:The constant region of amino acid sequence shown in 4 (Fc)。